Laboratory Biosafety Manual-WHO

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WHO/CDS/CSR/LYO/2003.4
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LABORATORY
BIOSAFETY MANUAL
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SECOND EDITION (REVISED)
WORLD HEALTH ORGANIZATION

Geneva 2003
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World Health Organization 2003
This publication is not a formal publication of the World Health Organization (WHO), and all rights are
reserved by the Organization. The publication may, however, be freely reviewed, abstracted, reproduced
and translated, in part or in whole, but not for sale or for use in conjunction with commercial purposes.
The views expressed in publications by named authors are solely the responsibility of those authors.
The designations employed and the presentation of the material in this publication, including tables and
maps, do not imply the expression of any opinion whatsoever on the part of the secretariat of the World
Health Organization concerning the legal status of any country, territory, city or area or of its authorities,
or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate
border lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers' products does not imply that they are
endorsed or recommended by WHO in preference to others of a similar nature that are not mentioned.
Errors and omissions excepted, the names of proprietary products are distinguished by initial capital
letters.
Contents
Foreword ...................................................................................................................................................... v
Acknowledgements .................................................................................................................................. vii
1.
General principles............................................................................................................................... 1
PART I Guidelines
Risk assessment................................................................................................................................. 7
Specimens for which there is limited information .............................................................................. 7
Risk assessment and genetically modified microorganisms ............................................................. 8
3.
Basic laboratories Biosafety Levels 1 and 2............................................................................... 10

Code of practice............................................................................................................................... 10
Laboratory design and facilities ....................................................................................................... 11
Laboratory equipment ...................................................................................................................... 13
Health and medical surveillance ...................................................................................................... 13
Training ............................................................................................................................................ 14
Waste handling ................................................................................................................................ 14
Chemical, fire, electrical and radiation safety .................................................................................. 16
4.
The containment laboratory Biosafety Level 3 ........................................................................... 17

Code of practice............................................................................................................................... 17
Health and medical surveillance ...................................................................................................... 18
5.
The maximum containment laboratory Biosafety Level 4 ......................................................... 20

Laboratory biosafety manual ........................................................................................................... 21
6.
Laboratory animal facilities ............................................................................................................. 22

Animal facility Biosafety Level 1 ................................................................................................... 22
Invertebrates .................................................................................................................................... 24
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2.
PART II Laboratory equipment

7.
Biological safety cabinets................................................................................................................ 29

Class I biological safety cabinet....................................................................................................... 30
Class II biological safety cabinets.................................................................................................... 30
Class III biological safety cabinet..................................................................................................... 32
Biological safety cabinet air connections ......................................................................................... 33
Selection of a biological safety cabinet............................................................................................ 33
Using biological safety cabinets in the laboratory............................................................................ 34
8.
Equipment-related hazards.............................................................................................................. 37
Equipment that may create a hazard............................................................................................... 37
Laboratory Biosafety Manual, 2nd revised edition
9.
Equipment designed to reduce biological hazards....................................................................... 39

Negative-pressure flexible-film isolators .......................................................................................... 40
Pipetting aids ................................................................................................................................... 40
Homogenizers, shakers, blenders and sonicators........................................................................... 41
Disposable transfer loops ................................................................................................................ 41
Microincinerators.............................................................................................................................. 41
Personal protective clothing and equipment.................................................................................... 41
PART III Good microbiological technique
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10. Safe laboratory techniques.............................................................................................................. 45

Safe handling of specimens in the laboratory.................................................................................. 45
Use of pipettes and pipetting aids.................................................................................................... 45
Avoiding the dispersal of infectious materials.................................................................................. 46
Use of biological safety cabinets ..................................................................................................... 46
Avoiding ingestion of infectious materials and contact with skin and eyes ..................................... 46
Avoiding injection of infectious materials ......................................................................................... 47
Separation of serum ........................................................................................................................ 47
Use of centrifuges ............................................................................................................................ 47
Use of homogenizers, shakers, blenders and sonicators................................................................ 48
Use of tissue grinders ...................................................................................................................... 48
Care and use of refrigerators and freezers...................................................................................... 48
Opening of ampoules containing lyophilized infectious materials ................................................... 48
Storage of ampoules containing infectious materials ...................................................................... 49
Special precautions with blood and other body fluids, tissues and excreta .................................... 49
Precautions with materials that may contain prions ........................................................................ 50
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11. Biosafety and recombinant DNA technology................................................................................. 52

Biological expression systems......................................................................................................... 52
Properties of the donor organism and cloned DNA ......................................................................... 52
Viral vectors for gene transfer.......................................................................................................... 53
Transgenic and knock-out animals................................................................................................ 53
Transgenic plants............................................................................................................................. 53
Conclusions ..................................................................................................................................... 53
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12. Transport of infectious substances................................................................................................ 55
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13. Contingency plans and emergency procedures............................................................................ 56

Contingency plan ............................................................................................................................. 56
Emergency procedures for microbiological laboratories.................................................................. 56
14. Disinfection and sterilization ........................................................................................................... 59
Definitions ........................................................................................................................................ 59
Precleaning and cleaning laboratory materials................................................................................ 59
Chemical germicides........................................................................................................................ 60
Local environmental decontamination ............................................................................................. 63
Decontamination of biological safety cabinets................................................................................. 63
Hand-washing/hand decontamination ............................................................................................. 64
Heat disinfection and sterilization .................................................................................................... 64
Incineration ...................................................................................................................................... 66
Disposal ........................................................................................................................................... 66
Decontamination of prion-containing materials ............................................................................... 66
Summary.......................................................................................................................................... 66
PART IV Chemical, fire and electrical safety
15. Hazardous chemicals ....................................................................................................................... 69
Definitions and classifications.......................................................................................................... 69
Routes of exposure.......................................................................................................................... 69
Storage of chemicals ....................................................................................................................... 69
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Contents
General rules ................................................................................................................................... 70

Toxic effects of chemicals................................................................................................................ 70
Explosive chemicals......................................................................................................................... 72
Chemical spillage............................................................................................................................. 72
Compressed and liquefied gases .................................................................................................... 73
16. Fire in the laboratory ........................................................................................................................ 74
17. Electrical hazards ............................................................................................................................. 75
PART V Safety organization and training
18. The biosafety officer and safety committee................................................................................... 77
Biosafety officer ............................................................................................................................... 77
Safety committee ............................................................................................................................. 78
General organization........................................................................................................................ 78
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19. Safety rules for support staff........................................................................................................... 79

Engineering and building maintenance services ............................................................................. 79
Cleaning (domestic) services........................................................................................................... 79
Safety rules for domestic and cleaning staff.................................................................................... 79
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20. Training programmes ....................................................................................................................... 81

Basic course: Good laboratory practice (GLP) ................................................................................ 82
Module 1 (the core module): Good microbiological technique (GMT)............................................. 83
Module 2: The safe laboratory environment .................................................................................... 83
Module 3: GLP for support staff....................................................................................................... 84
Module 4: GLP for safety staff ......................................................................................................... 84
Module 5: GLP for specialist staff who handle microorganisms in Risk Groups 3 and 4 ................ 85
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21. Safety checklist ................................................................................................................................. 87

Laboratory premises ........................................................................................................................ 89
Storage facilities............................................................................................................................... 89
Sanitation and staff facilities ............................................................................................................ 89
Heating and ventilation .................................................................................................................... 89
Lighting ............................................................................................................................................ 90
Services ........................................................................................................................................... 90
Security ............................................................................................................................................ 90
Fire prevention ................................................................................................................................. 90
Flammable liquid storage................................................................................................................. 90
Electrical hazards............................................................................................................................. 91
Compressed and liquefied gases .................................................................................................... 91
Personal protection .......................................................................................................................... 91
Health and safety of staff ................................................................................................................. 91
Infectious materials .......................................................................................................................... 92
Chemicals and radioactive substances ........................................................................................... 92
References ................................................................................................................................................. 94
ANNEX 1 Immunization of staff ............................................................................................................... 98
ANNEX 2 WHO Biosafety Collaborating Centres................................................................................... 99
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Foreword
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The World Health Organization (WHO) has long recognized that safety and, in particular, biological safety
are important international issues. This specialized agency of the United Nations published the first
edition of its Laboratory biosafety manual in 1983. The manual encourages countries to prepare specific
codes of practice for the safe handling of pathogenic microorganisms in laboratories within their
geographical borders, and provides expert guidance for developing such codes of practice.
This web-based revision of the second edition is the first step in the preparation of a third edition,
planned for publication in 2003. The new publication will combine the Laboratory biosafety manual, 2nd
1
ed. (revised) and the contents of the document entitled Safety in health-care laboratories .
The authors and editor of this revised second edition continue the now-established WHO tradition of
promoting health and safety in microbiological laboratories in the international community. While the
technical content affirms the guidance of previous editions, there are helpful changes in the organization
of the text and valuable new material is included. The importance of personal responsibility for safe
laboratory activities is stressed throughout the manual. A safe and healthful laboratory environment is the
product of individuals who are well trained and technically proficient in safe practices, and share
responsibility for their own safety and for the safety of their colleagues, their communities and the
environment. Personal responsibility also involves the practice of assessing risks prior to the conduct of
activities that involve new protocols or new pathogens. Two new chapters address risk assessment and
recombinant DNA technology. These timely additions provide thoughtful and concise guidance for
assessing risks in the contemporary microbiology laboratory.
A safe and healthful laboratory environment is also the product of responsible institutional leadership.
National codes of practice foster and promote good institutional leadership in biosafety. The revised
second edition of the WHO Laboratory biosafety manual is, like the previous editions, a helpful reference
and guide to nations that accept the challenge to develop national codes of practice.
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W. Emmett Barkley, PhD

Director, Office of Laboratory Safety
Howard Hughes Medical Institute
Chevy Chase, MD, USA
Safety in health-care laboratories. Geneva, World Health Organization, 1999 (unpublished document WHO/LAB/97.1). Obtainable
on request from Department of Vaccines and Other Biologicals, World Health Organization, 1211 Geneva 27, Switzerland;
http://www.who.int/gpv-documents/.
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Acknowledgements
The development of this revised second edition of the Laboratory biosafety manual has been made
possible through the contributions of the following, whose expertise is gratefully acknowledged:
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Dr Ingegerd Kallings, Swedish Institute of Infectious Disease Control, Stockholm, Sweden

Ms Mary Ellen Kennedy, Consultant in Biosafety, Ashton, Ontario, Canada (Technical editor)
Ms Margery Kennett, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia
The late Dr Richard Knudsen, Office of Health and Safety, Centers for Disease Control and
Prevention, Atlanta, GA, USA
Dr Nicoletta Previsani, Biosafety Programme, World Health Organization, Geneva, Switzerland
Dr Jonathan Richmond, Office of Health and Safety, Centers for Disease Control and Prevention,
Atlanta, GA, USA
Dr Syed A. Sattar, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada
Dr Deborah E. Wilson, Division of Safety, National Institutes of Health, Washington, DC, USA
Dr Riccardo Wittek, Institute of Animal Biology, University of Lausanne, Lausanne, Switzerland
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1. General principles
Throughout this manual, references are made to the relative hazards of infective microorganisms by risk
group (WHO Risk Groups 1, 2, 3 and 4). This risk group classification is to be used for laboratory work
only. Laboratories are designated according to their design features, construction and containment
facilities as basic Biosafety Level 1, basic Biosafety Level 2, containment Biosafety Level 3 and
maximum containment Biosafety Level 4. Table 1 describes the risk groups, Table 2 relates them to the
laboratory designations, and Table 3 summarizes the requirements at the four biosafety levels.
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Risk Group 2 (moderate individual risk, low community risk)
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A microorganism that is unlikely to cause human or animal disease.
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Risk Group 1 (no or very low individual and community risk)
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Table 1. Classification of infective microorganisms by risk group
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A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to
laboratory workers, the community, livestock or the environment. Laboratory exposures may cause
serious infection, but effective treatment and preventive measures are available and the risk of spread
of infection is limited.
Risk Group 3 (high individual risk, low community risk)
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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.
Risk Group 4 (high individual and community risk)
A pathogen that usually causes serious human or animal disease and that can be readily transmitted
from one individual to another, directly or indirectly. Effective treatment and preventive measures are
not usually available.
a
The concept and classification of risk groups are being reevaluated and will be addressed in the third edition of the Laboratory
biosafety manual.
Table 2. Relation of risk groups to biosafety levels, practices and equipment

Risk
Group
1
2
Biosafety Level
Laboratory type
Laboratory practices
Safety equipment
Basic Biosafety
Level 1
Basic teaching,
research
GMT
None; open bench

work
Basic Biosafety
Level 2
Primary health
services;
diagnostic,
research
GMT plus protective

clothing, biohazard
sign
Open bench plus

BSC for potential
aerosols
Risk
Group
3
Biosafety Level
Laboratory type
Laboratory practices
Safety equipment
Containment Biosafety
Level 3
Special
diagnostic,
research
As Level 2 plus special

clothing, controlled
access, directional air
flow
BSC and/or other

primary devices for
all activities
Maximum containment
Biosafety Level 4
Dangerous
pathogen units
As Level 3 plus airlock

entry, shower exit,
special waste disposal
Class III BSC, or

positive pressure
suits in conjunction
with Class II BSCs,
double-ended
autoclave (through
the wall), filtered air
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BSC, biological safety cabinet; GMT, good microbiological technique
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Pathogenicity of the organism.

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.
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.
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.
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Countries (regions) should draw up a national (regional) classification of microorganisms, by risk

group, based on the following factors.
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Table 3. Summary of biosafety level requirements
Isolation of laboratory
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Biosafety level
Room sealable for decontamination
No
No
Desirable
Yes
No
No
Yes
Yes
Ventilation:
inward air flow
No
Desirable
Yes
Yes
mechanical via building system
No
Desirable
Yes
No
mechanical, independent
No
Desirable
Yes
Yes
HEPA filtered air exhaust
No
No
Desirable
Yes
Double-door entry
No
No
Yes
Yes
Airlock
No
No
No
Yes
Airlock with shower
No
No
No
Yes
Anteroom
No
No
Yes
No
Anteroom with shower
No
No
Desirable
No
Effluent treatment
No
No
Desirable
Yes
General principles
Biosafety level
1
Autoclave:
on site
Yes
Yes
Yes
Yes
in laboratory room
No
No
Desirable
Yes
double-ended
No
No
Desirable
Yes
Biological safety cabinets:

Class I
No
Optional
Yes
No
Class II
No
Desirable
Yes
Yes, in
conjunction
with suit
laboratories
Class Ill
No
No
Desirable
Yes, in
conjunction
with cabinet
laboratories
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In assessing the various criteria for classification, it is important to take into account conditions
prevailing in the geographical area in which the microorganisms are handled.
In the preparation of classification lists it is recommended that, where appropriate, some additional
information is given about the advisability of the use of personal protective equipment and primary
containment devices (e.g. biological safety cabinets). Consideration must also be given to enhancing
biosafety practices and procedures and general containment levels for organisms known to be multidrugresistant and in cases where high volumes or concentrations of an agent may be used. Examples of
classification lists are available from the Centers for Disease Control and Prevention (CDC) and the
National Institute of Health (NIH), USA (1), and the European Union (EU) (2).
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PART I
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Guidelines
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2. Risk assessment
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pathogenicity of the agent and infectious dose

consideration of the outcome of exposure
natural route of infection
other routes of infection, resulting from laboratory manipulations (parenteral, airborne, ingestion)
stability of the agent in the environment
concentration of the agent and volume of concentrated material to be manipulated
presence of a suitable host (human or animal)
information available from animal studies and reports of laboratory-acquired infections or clinical
reports
laboratory activity planned (concentration, sonication, aerosolization, centrifugation, etc.)
any genetic manipulation of the organism that may extend the host range of the agent or alter the
agents sensitivity to known, effective treatment regimens (see Risk assessment and genetically
modified microorganisms, below)
local availability of effective prophylaxis or therapeutic interventions.
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The backbone of the practice of biosafety is risk assessment. While there are many tools available to
assist in the assessment of risk for a given procedure or experiment, the most important component is
professional judgement. Risk assessments should be performed by the individuals most familiar with the
specific characteristics of the organisms being considered for use, the equipment and procedures to be
employed, animal models that may be used, and the containment equipment and facilities available. The
laboratory director or principal investigator is responsible for ensuring that adequate and timely risk
assessments are performed and for working closely with the institutions safety committee (if existing)
and biosafety personnel (if existing) to ensure that appropriate equipment and facilities are available to
support the work being considered. Once performed, risk assessments should be routinely reviewed and
revised when necessary, taking into consideration acquisition of new data having a bearing on the degree
of risk and other relevant new information from the scientific literature.
One of the most helpful tools available for performing a microbiological risk assessment is the listing of
risk groups for microbiological agents (see Chapter 1). However, simple reference to the risk grouping for
a particular agent is insufficient in the conduct of a risk assessment. Other factors that should be
considered, as appropriate, include:
On the basis of the information ascertained during the risk assessment, a biosafety level can be
assigned to the planned work and appropriate personal protective equipment selected.
Specimens for which there is limited information

The risk assessment procedure described above works well when there is adequate information
available. However, there are situations when the information is insufficient to perform an appropriate risk
assessment, for example, with clinical specimens or epidemiological samples collected in the field. In
these cases, it is prudent to take a conservative approach to specimen manipulation.
Universal precautions (3) should always be followed, and barrier protections applied (gloves, gowns,
eye protection), regardless of the origin of the samples.
Basic containment Biosafety Level 2 should be the minimum requirement for the handling of
specimens.
Transport of specimens should follow national and/or international rules and regulations.
Some information may be available to assist in determining the risk of handling these specimens:
medical data on the patient
.
.
epidemiological data (morbidity and mortality data, suspected route of transmission, other outbreak
investigation data)
information on the geographical origin of the specimen.
Risk assessment and genetically modified microorganisms
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Risk assessment for work with genetically modified microorganisms (GMMOs) must include an
assessment of the degree of potential harm that may arise, the severity of consequence, and the
likelihood or frequency of that harm occurring. It must be suitable and sufficient; it need not necessarily
be very detailed. It may be simple, for example, where it is immediately obvious that the risks are low or
that the proposed control measures are clearly adequate. For an operation involving a low-hazard, wellknown and well-understood organism, it may be possible to determine the outcome of the assessment
almost at first glance. However, for a complex operation involving more hazardous organisms about
which there may be some degree of uncertainty, the assessment will have to be extensive and may
necessitate the acquisition of new data.
Potential hazards (harmful effects) of work involving GMMOs may be associated with the primary
recipient or host organism, other potential recipients in the environment, or the donor microorganism. In
many cases, the characteristics of the host organism will be more relevant to the risk assessment than
those of the donor organism. Factors to consider during hazard identification include pathogenicity, the
biological activity or toxicity of the foreign gene product, and the mobility of plasmid or viral vectors. As a
general guide, if a donor organism is used only as a source of well-characterized DNA for a selectable
phenotype (e.g. kanamycin resistance or -galactosidase activity) or a promotor or other control
sequence, the characteristics of the donor will not need to be considered. If, however, the insert contains
genes that encode biologically active molecules, toxins or virulence factors, then relevant information
from the donor organism should be considered.
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Hazards associated with the host/recipient

Consideration should be given to:
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susceptibility of the host

pathogenicity of the host strain, including virulence, infectivity, toxin production and modification of
the host range
degree of immunity of the recipient and status of immune system
seriousness of the consequences of exposure.
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Hazards arising directly from the inserted gene

Assessment is necessary in situations where the product of the inserted gene has known biologically
active properties that may give rise to harm, for example:
.
.
.
.
.
.
.
toxins
cytokines
hormones
gene expression regulators
virulence factors or enhancers
antibiotic resistances
allergens.
The consideration of such cases should include an estimation of the level of expression required to
achieve biological activity.
Hazards arising from the alteration of existing pathogenic traits

Many modifications do not involve genes whose products are inherently harmful but adverse effects may
arise as the result of alteration of existing non-pathogenic or pathogenic traits. Modification of normal
genes may alter pathogenicity. In an attempt to identify these potential hazards, the following points may
be considered (the list is not exhaustive).
.
.
.
.
.
.
.
Risk assessment
Is there an increase in infectivity or pathogenicity?

Could any disabling mutation within the recipient be overcome as a result of the insertion of the
foreign gene?
Does the foreign gene encode a pathogenicity determinant from another organism (bacterial toxins,
invasins, integrins and surface structures such as membrane glycoproteins and
lipopolysaccharides)?
If the foreign DNA does include a pathogenicity determinant, is it foreseeable that this gene could
contribute to the pathogenicity of the GMMO?
Is treatment available?
Will the susceptibility of the GMMO to antibiotics or other forms of therapy be affected as a
consequence of the genetic modification?
Is eradication of the GMMO achievable?
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For further information see Chapter 11, and references 4 and 5.
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Other factors that need to be considered in the GMMO risk assessment are the presence of any
potentially oncogenic gene sequences and potential hazards associated with the cell lines being used,
such as the presence of adventitious agents. The introduction of whole animals or plants into an
experiment also requires careful consideration. The investigator must be familiar and comply with the
regulations, restrictions and requirements for the conduct of work with GMMOs at his or her institution.
3. Basic laboratories Biosafety Levels 1 and 2
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For the purposes of this manual, the guidance and recommendations given as minimum requirements
pertaining to laboratories of all biosafety levels are directed at microorganisms in Risk Groups 14.
Although some of the precautions may appear to be unnecessary for some organisms in Risk Group 1,
they are desirable for training purposes to promote good (i.e. safe) microbiological techniques.
Diagnostic and health care laboratories (public health, clinical or hospital-based) must all be designed
for Biosafety Level 2 or above. As no laboratory has complete control over the specimens it receives,
laboratory workers may occasionally and unexpectedly be exposed to organisms in higher risk groups
than anticipated. This possibility must be recognized in the development of safety plans and policies. In
some countries, accreditation of clinical laboratories is required. Globally, universal precautions (3)
should always be adopted and practised.
The guidelines for basic laboratories Biosafety Levels 1 and 2 presented here are comprehensive
and detailed, as they are fundamental to all levels of laboratories. The guidelines for containment
laboratories Biosafety Level 3 and maximum containment laboratories Biosafety Level 4 that follow
(Chapters 4 and 5) are modifications of and additions to these guidelines, designed for work with the
more dangerous (hazardous) pathogens.
Code of practice
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This code is a listing of the most essential laboratory practices and procedures that are basic to good
microbiological techniques. In many laboratories and national laboratory programmes, this code may be
used to develop written practices and procedures for safe laboratory operations.
Each laboratory should adopt a safety or operations manual that identifies known and potential
hazards, and specifies practices and procedures to eliminate or minimize such hazards. Good
microbiological technique is fundamental to laboratory safety. Specialized laboratory equipment is a
supplement to but can never replace appropriate procedures. The most important concepts are listed
below.
1. The international biohazard warning symbol and sign (Fig. 1) 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 under the age of 16 years should not be authorized or allowed to enter laboratory working
areas.
5. Access to animal houses should be specially authorized.
6. Animals not involved in the work of the laboratory should not be permitted in the laboratory.
7. No smoking No eating and No drinking signs should be displayed clearly inside and outside the
laboratory.
Personal protection
1. Laboratory coveralls, gowns or uniforms must be worn at all times for work in the laboratory.
2. Appropriate gloves must be worn for all procedures that may involve direct or accidental contact with
blood, infectious materials or infected animals. After use, gloves should be removed aseptically and
hands must then be washed.
3. Personnel must wash their hands after handling infectious materials and animals, and before they
leave the laboratory working areas.
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Basic laboratories Biosafety Levels 1 and 2
4. Safety glasses, face shields (visors) or other protective devices must be worn when it is necessary to
protect the eyes and face from splashes, impacting objects and sources of artificial ultraviolet
radiation.
5. It is prohibited to wear protective laboratory clothing outside of the laboratory, e.g. in canteens, coffee
rooms, offices, libraries, staff rooms and toilets.
6. Open-toed footwear should not be worn in laboratories.
7. Eating, drinking, applying cosmetics and handling contact lenses is prohibited in the laboratory
working areas.
8. Storing human foods or drinks anywhere in the laboratory working areas is prohibited.
9. Protective laboratory clothing should not be stored in the same lockers or cupboards as street
clothing.
Procedures
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1. Pipetting by mouth must be strictly forbidden.

2. Materials must not be placed in the mouth. Labels must not be licked.
3. All technical procedures should be performed in a way that minimizes the formation of aerosols and
droplets. Whenever there is an increased risk of aerosolization, work should be conducted in a
biological safety cabinet.
4. The use of hypodermic needles and syringes should be limited. They must not be used as substitutes
for pipetting devices or for any purpose other than parenteral injection or aspiration of fluids from
laboratory animals.
5. All spills, accidents and overt or potential exposures to infectious materials must be reported to the
laboratory supervisor. A written record of such accidents and incidents should be maintained.
6. A written procedure for the clean up of all spills must be developed and followed.
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Laboratory working areas
Biosafety management
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1. The laboratory should be kept neat, clean and free of materials that are not pertinent to the work.
2. Work surfaces must be decontaminated after any spill of potentially dangerous material and at the
end of the working day.
3. All contaminated materials, specimens and cultures must be decontaminated before disposal or
cleaning for reuse.
4. Packing and transportation must follow applicable national and/or international regulations.
5. When windows can be opened, they should be fitted with arthropod-proof screens.
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1. It is the responsibility of the laboratory director (the person who has immediate responsibility for the
laboratory) to ensure the development and adoption of a biosafety management plan and a safety or
operations manual.
2. The laboratory supervisor (reporting to the laboratory director) should ensure that regular training in
laboratory safety is provided.
3. Personnel should be advised of special hazards and required to read the safety or operations manual
and follow standard practices and procedures. The laboratory supervisor should make sure that all
personnel understand these. A copy of the safety or operations manual should be available in the
laboratory.
4. When appropriate, there should be an arthropod and rodent control programme.
5. Appropriate medical evaluation, surveillance and treatment should be provided for all personnel in
case of need, and adequate medical records should be maintained.
6. Baseline serum samples may be collected from laboratory staff and other persons at risk. These
should be stored appropriately according to national or local guidelines. Additional specimens mayu
be collected periodically depending on the organisms handled and the function of the laboratory.
Laboratory design and facilities

In designing a laboratory and assigning certain types of work to it, special attention should be paid to
conditions that are known to pose safety problems. These include:
11
formation of aerosols
work with large volumes and/or high concentrations of microorganisms
overcrowding and too much equipment
infestation with rodents and arthropods
unauthorized entrance
workflow: use of specific samples and reagents.
Design features
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1. Ample space must be provided for the safe conduct of laboratory work and for cleaning and
maintenance.
2. Walls, ceilings and floors should be smooth, easy to clean, impermeable to liquids and resistant to
the chemicals and disinfectants normally used in the laboratory. Floors should be slip-resistant.
Exposed pipes and ducting should be avoided where possible.
3. Bench tops should be sealed to the walls, impervious to water and resistant to disinfectants, acids,
alkalis, organic solvents and moderate heat.
4. Illumination should be adequate for all activities. Undesirable reflections and glare should be avoided.
5. Laboratory furniture should be sturdy. Open spaces between and under benches, cabinets and
equipment should be accessible for cleaning.
6. Storage space must be adequate to hold supplies for immediate use and thus prevent clutter on
bench tops and in aisles. Additional long-term storage space, conveniently located outside the
laboratory working areas, should also be provided.
7. Space and facilities should be provided for the safe handling and storage of solvents, radioactive
materials, and compressed and liquefied gases.
8. Facilities for storing outer garments and personal items should be provided outside the laboratory
working areas.
9. Facilities for eating and drinking and for rest should be provided outside the laboratory working areas.
10. Hand-wash basins, with running water if possible, should be provided in each laboratory room,
preferably near the exit door.
11. Doors should have vision panels, be self-closing and have appropriate fire ratings.
12. An autoclave should be available in the same building as the laboratory.
13. Safety systems should cover fire, electrical emergencies, emergency shower and eyewash facilities.
14. First-aid areas or rooms suitably equipped and readily accessible should be available.
15. There are no specific ventilation requirements for laboratories handling Risk Group 1 and 2
microorganisms. However, in the planning of new facilities, consideration should be given to the
provision of mechanical ventilation systems that provide an inward flow of air without recirculation. If
there is no mechanical ventilation, windows should be able to be opened and should be fitted with
arthropod-proof screens.
16. A dependable supply of good quality water is essential. There should be no cross-connections
between sources of laboratory and drinking-water supplies. An anti-backflow device should protect
the public water system.
17. There should be a reliable and adequate electricity supply and emergency lighting to permit safe exit.
A stand-by generator is desirable for the support of essential equipment, such as incubators,
biological safety cabinets, freezers, etc., and for the ventilation of animal cages.
18. There should be a reliable and adequate supply of gas. Good maintenance of the installation is
mandatory.
19. Three aspects of waste disposal need special attention to meet performance and pollution-control
requirements:
autoclaves for the treatment of solid waste need specially designed accommodation and services
incinerators should be of special design, equipped with afterburners and smoke-consuming
devices
contaminated wastewater must be decontaminated.
20. Laboratories and animal houses are occasionally the targets of vandals. Physical and fire security
must be considered. Strong doors, screened windows, and restricted issue of keys are compulsory.
Other measures should be considered and applied, as appropriate, to augment security.
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Laboratory equipment
Together with good procedures and practices, the use of safety equipment will help to reduce risks when
dealing with biosafety hazards. This section deals with basic principles related to equipment suitable for
laboratories of all biosafety levels. Requirements for laboratory equipment pertinent to higher biosafety
levels are dealt with in the relevant chapters.
The laboratory director should, after consultation with the biosafety officer and safety committee (if
designated), ensure that adequate equipment is provided and that it is used properly. Equipment should
be selected to take account of certain general principles, i.e. it should be:
designed to prevent or limit contact between the operator and the infectious material
constructed of materials that are impermeable to liquids, resistant to corrosion and meet structural
requirements
fabricated to be free of burrs, sharp edges and unguarded moving parts
designed, constructed and installed to facilitate simple operation and provide for ease of
maintenance, cleaning, decontamination and certification testing; glassware and other breakable
materials should be avoided, whenever possible.
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Detailed performance and construction specifications may need to be consulted to ensure that the
equipment possesses the necessary safety features.
Essential biosafety equipment
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1. Pipetting aids to avoid mouth pipetting. Many different designs are available.
2. Biological safety cabinets, to be used whenever:
infectious materials are handled; such materials may be centrifuged in the open laboratory if
sealed centrifuge safety cups are used and if they are loaded and unloaded in a biological safety
cabinet
there is an increased risk of airborne infection
procedures with a high potential for producing aerosols are used; these may include
centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening of
containers of infectious materials whose internal pressure may be different from the ambient
pressure, intranasal inoculation of animals, and harvesting of infectious tissues from animals and
eggs.
3. Plastic disposable transfer loops. Alternatively, electric transfer loop incinerators may be used inside
the biological safety cabinet (BSC) to reduce aerosol production.
4. Screw-capped tubes and bottles.
5. Autoclaves to decontaminate infectious materials.
6. Plastic disposable Pasteur pipettes, whenever available, to avoid glass.
7. Equipment such as autoclaves and biological safety cabinets must be validated with appropriate
methods (usually by a certified examiner) before being taken into use. Recertification should take
place at regular intervals, according to the manufacturers instructions.
Health and medical surveillance

The employing authority, through the laboratory director, is responsible for ensuring that there is
adequate surveillance of the health of laboratory personnel. The objective of such surveillance is to
monitor for occupationally acquired diseases. Appropriate activities to achieve these objectives are to:
provide active or passive immunization where indicated (see Annex 1)
facilitate the early detection of laboratory-acquired infections
exclude highly susceptible individuals (such as pregnant women) from highly hazardous laboratory
work
provide effective personal protective equipment and procedures.
Guidelines for the surveillance of laboratory workers handling microorganisms in Risk

Group 1
Historical evidence indicates that these microorganisms are unlikely to cause human disease or animal
disease of veterinary importance. Ideally, however, all laboratory workers should undergo a pre-
13
employment health check at which their medical history is recorded. Prompt reporting of illnesses or
laboratory accidents is desirable and all staff members should be made aware of the importance of
maintaining good microbiological techniques.
Guidelines for the surveillance of laboratory workers handling microorganisms in Risk

Group 2
1. A pre-employment or preplacement health check is necessary. The persons medical history should
be recorded and a clinical examination and collection of baseline serum sample are recommended.
2. Records of illness and absence should be kept by the laboratory management; it is the responsibility
of the laboratory worker and his or her own medical adviser to keep the laboratory director informed
of all absences due to illness.
3. Women of childbearing age should be made aware of the risk to an unborn child of occupational
exposure to certain microorganisms, e.g. rubella virus. The precise steps taken to protect the fetus
will vary, depending on the microorganisms to which the women may be exposed.
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Training
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Human error and poor technique can compromise the best of safeguards to protect the laboratory worker.
Thus, a safety-conscious staff, well informed about the recognition and control of laboratory hazards, is
key to the prevention of laboratory-acquired infections, incidents and accidents. For this reason,
continuous in-service training in safety measures is essential. An effective safety programme begins with
the laboratory managers, who should ensure that safe laboratory practices and procedures are integrated
into the basic training of employees. Training in safety measures should be an integral part of new
employees introduction to the laboratory. Employees should be introduced to the code of practice and to
local guidelines. Measures to assure that employees have read and understood the guidelines, such as
signature pages, should be adopted. Laboratory supervisors play the key role in training their immediate
staff in good laboratory techniques. The biosafety officer can assist in training and with the development
of training aids and documentation.
Staff training should always include safe methods for dealing with the following highly hazardous
procedures that are commonly encountered by all laboratory personnel, involving:
inhalation risks (i.e. aerosol production), such as using loops, streaking agar plates, pipetting, making
smears, opening cultures, taking blood/serum samples, centrifugation
ingestion risks, such as handling specimens, smears and cultures
risks of percutaneous exposures, through the use of syringe and needle techniques
animal handling that may result in bites and scratches
handling of blood and other potentially hazardous pathological materials
decontamination and disposal of infectious material.
Waste handling
Waste is anything that is to be discarded.
In laboratories, decontamination of wastes and their ultimate disposal are closely interrelated. In terms
of daily use, few if any contaminated materials will require actual removal from the laboratory or
destruction. Most glassware, instruments and laboratory clothing will be reused or recycled. The
overriding principle is that all infectious materials should be decontaminated, autoclaved or incinerated
within the laboratory.
The principal questions to be asked before discharge of any objects or materials from laboratories that
deal with potentially infectious microorganisms or animal tissues are as follows.
1. Have the objects or materials been effectively decontaminated or disinfected by an approved
procedure?
2. If not, have they been packaged in an approved manner for immediate on-site incineration or transfer
to another facility with incineration capacity?
3. Does the disposal of the decontaminated objects or materials involve any additional potential
hazards, biological or otherwise, to those who carry out the immediate disposal procedures or who
might come into contact with discarded items outside the facility?
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Decontamination
Steam autoclaving is the preferred method for all decontamination processes. Materials for
decontamination and disposal should be placed in containers, e.g. autoclavable plastic bags that are
colour-coded according to whether the contents are to be autoclaved and/or incinerated. Alternative
methods may be envisaged only if they remove and/or kill microorganisms (for more details see
Chapter 14).
Disinfectants and chemicals
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The safety or operations manual should include a written policy stating which disinfectants are to be used
for what purposes, and the manufacturers recommended dilution for each disinfectant. The manufacturer
should be able to provide relevant documentation. Make sure each disinfectant has been validated for its
indicated use in the laboratory.
Sodium hypochlorite and phenolic compounds are the disinfectants recommended for general
laboratory use.
For special purposes, various surface-active or lipid-destroying agents, including alcohols, iodine,
iodophors and other oxidizing agents, as well as a very high or extremely low pH, can be effective,
provided that it has been established that the agent to be destroyed is not resistant to the procedure.
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Other methods
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Waste handling and disposal procedures
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The use of dry heat is discouraged because of its unpredictable variation. Similarly, microwave, ultraviolet
and ionizing radiation are unsuitable.
New technologies, including alkaline hydrolysis, may be used as a substitute for incineration for
terminal infectious waste treatment.
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An identification and separation system for infectious materials and their containers should be adopted.
Categories should include the following.
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1. Non-contaminated (non-infectious) waste that can be reused or recycled or disposed of as general,

household waste.
2. Contaminated (infectious) sharps hypodermic needles, scalpels, knives and broken glass; these
should always be collected in puncture-proof containers fitted with covers and treated as infectious.
3. Contaminated material for decontamination by autoclaving and thereafter washing and reuse or
recycling.
4. Contaminated material for autoclaving and disposal.
5. Contaminated material for direct incineration.
Sharps
After use, hypodermic needles should not be recapped, clipped or removed from disposable syringes.
The complete assembly should be placed in a sharps container. Sharps containers must be puncture
proof and not be filled to capacity. When they are three-quarters full they should be placed in infectious
waste containers and incinerated, with prior autoclaving if laboratory practice requires it. Sharps
containers must not be disposed of in landfills.
Disposable syringes, used alone or with needles, should be placed in containers and incinerated, with
prior autoclaving if required.
Contaminated (infectious) materials for autoclaving and reuse

No precleaning should be attempted of any contaminated (infectious) materials to be autoclaved and
reused. Any necessary cleaning or repair must be done only after autoclaving or disinfection.
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Contaminated (infectious) materials for disposal
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Apart from sharps, which are dealt with above, all contaminated (potentially infectious) materials should
be autoclaved in leakproof containers, e.g. autoclavable, colour-coded plastic bags, before disposal. After
autoclaving, the material may be placed in transfer containers for transport to the incinerator. If possible,
materials deriving from health care activities should not be discarded in landfills even after
decontamination. If an incinerator is available on the laboratory site, autoclaving may be omitted: the
contaminated waste should be placed in designated containers (e.g. colour-coded bags) and transported
directly to the autoclave or incinerator. Reusable transfer containers should be leakproof and have tightfitting covers. They should be disinfected and cleaned before they are returned to the laboratory for
further use.
Discard pots, pans or jars, preferably unbreakable (e.g. plastic), and containing a suitable disinfectant,
freshly prepared each day, should be placed at every work station. Waste materials should remain in
intimate contact with the disinfectant (i.e. not protected by air bubbles) for the appropriate time, according
to the disinfectant used (see Chapter 14). The disinfectant should then be poured into a container for
autoclaving or incineration. The discard pots should be autoclaved and washed before reuse.
Incineration is the method of choice for the final disposal of contaminated waste, including carcasses
of laboratory animals (see section on Incineration in Chapter 14). Incineration of contaminated waste
must meet with the approval of the public health and air pollution authorities, as well as that of the
laboratory biosafety officer.
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Chemical, fire, electrical and radiation safety
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A breakdown in the containment of pathogenic organisms may be the indirect result of chemical, fire,
electrical or radiation accidents. It is therefore essential to maintain high standards of safety in these
fields in any microbiological laboratory. Statutory rules and regulations for each of these will normally be
laid down by the competent national or local authority, whose assistance should be sought if necessary.
Chemical, fire and electrical hazards are considered in greater detail in Part IV of this manual
(Chapters 1517).
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4. The containment laboratory

Biosafety Level 3
code of practice
laboratory design and facilities
health and medical surveillance.
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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. Biosafety Level 3 containment requires the strengthening of the
operational and safety programmes above those for basic laboratories Biosafety Levels 1 and 2 (set out
in Chapter 3).
The guidelines given in this chapter are presented in the form of additions to those for basic
laboratories Biosafety Levels 1 and 2, which must therefore be applied before those specific for the
containment laboratory Biosafety Level 3. The major additions and changes are in the following:
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Laboratories in this category should be registered or listed with the national or other appropriate
health authorities.
Code of practice
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The code of practice for basic laboratories Biosafety Levels 1 and 2 applies except where modified as
follows.
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1. The two-person rule should apply, whereby no individual ever works alone in the laboratory.
2. The international biohazard warning symbol and sign (see Fig. 1) displayed on laboratory access
doors must identify the microorganism(s) handled and the name of the laboratory supervisor who
controls access, and indicate any special conditions for entry into the area, e.g. immunization.
3. Laboratory protective clothing must be of the type with solid-front or wrap-around gowns, scrub suits,
coveralls, head covering and, where appropriate, shoe covers or dedicated shoes. Front-buttoned
standard laboratory coats are unsuitable. Laboratory protective clothing must not be worn outside the
laboratory, and it must be decontaminated before it is laundered.
4. When appropriate, respiratory equipment must be worn in rooms containing infected animals.

The containment laboratory Biosafety Level 3 is designed for work with Risk Group 3 microorganisms
and with large volumes and high concentrations of Risk Group 2 microorganisms, where there is a high
risk of aerosol spread and subsequent life-threatening consequences from infection.
The laboratory design and facilities for basic laboratories Biosafety Levels 1 and 2 apply except
where modified as follows.
1. The laboratory should be separated from the areas that are open to unrestricted traffic flow within the
building. Additional separation may be achieved by placing the laboratory at the blind end of a
corridor, or constructing a partition and door or access through an anteroom or basic laboratory
Biosafety Level 2.
2. Entry for personnel must be through a vestibule (i.e. double-door entry).
3. Access to the laboratory area must be designed to prevent entrance of arthropods and other vermin.
4. Access doors must be self-closing and interlockable. A break-through panel may be provided for
emergency exit use.
5. The surfaces of walls, floors and ceilings should be water-resistant and easy to clean. Openings in
these surfaces (e.g. for service pipes) should be sealed to facilitate decontamination of the room(s).
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6. The laboratory room must be sealable for decontamination. Air-ducting systems must be constructed
to permit gaseous decontamination.
7. Windows must be closed, sealed and break-resistant.
8. A foot- or elbow-operated or automatically controlled water source at the hand-wash basin should be
provided near to each exit door.
9. There must be a ventilation system that establishes a directional air flow from access spaces into the
laboratory room. Staff must at all times ensure that proper directional air flow into the laboratory room
is maintained.
10. The building ventilation system must be so constructed that air from the containment laboratory
Biosafety Level 3 is not recirculated to other areas within the building. Air may be HEPA filtered,
reconditioned and recirculated within that laboratory. Exhaust air from the laboratory (other than from
biological safety cabinets) must be discharged to the outside of the building, so that it is dispersed
away from occupied buildings and air intakes. It is recommended that this air is discharged through
high-efficiency particulate air (HEPA) filters.
11. Biological safety cabinets should be sited away from walking areas and out of cross-currents from
doors and ventilation systems (see Chapter 7).
12. The exhaust air from Class I or Class II biological safety cabinets (see Chapter 7), which will have
been passed through HEPA filters, must be discharged in such a way as to avoid interference with
the air balance of the cabinet or the building exhaust system. All HEPA filters must be installed in a
manner that permits gaseous decontamination and testing.
13. An autoclave for the decontamination of contaminated waste material should be available in the
containment laboratory. If infectious wastes have to be transported out of the containment laboratory
for disposal, they must be transported in sealed, unbreakable and leakproof containers according to
national or international regulations, as appropriate.
14. Anti-backflow devices must be fitted to the water supply.
15. Effluents should be decontaminated before being discharged to the sanitary sewer.
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The principles for the selection of equipment, including biological safety cabinets (see Chapter 7), are the
same as for the basic laboratory Biosafety Level 2, except that activities involving infectious materials
are conducted in biological safety cabinets whenever possible, together with other physical containment
devices, or special personal protective equipment. While Class I or Class II biological safety cabinets are
normally used in the containment laboratory Biosafety Level 3, a Class III biological safety cabinet may
be needed for high-risk procedures involving Risk Group 3 microorganisms, in accordance with local
national rules.
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Health and medical surveillance
The objectives of health and medical surveillance programmes for basic laboratories Biosafety Levels 1
and 2 also apply to containment laboratories Biosafety Level 3, except where modified as follows.
1. Medical examination of all laboratory personnel who work in Biosafety Level 3 containment
laboratories is mandatory. This should include recording of a detailed medical history and a physical
examination.
2. A baseline serum sample should be obtained and stored for future reference.
3. Individuals who are immunocompromised should not be employed in facilities with Biosafety Level 3
containment laboratories.
4. Special consideration should be given to the employment of pregnant women (see section on
Guidelines for the surveillance of laboratory workers handling microorganisms in Risk Group 2 in
Chapter 3).
5. After a satisfactory clinical assessment, the examinee should be provided with a medical contact card
(Fig. 2) stating that he or she is employed in a facility with a containment laboratory Biosafety
Level 3. It is suggested that this card should include a picture of the card holder, should be walletsized and should always be carried by the holder.
Note. The name(s) of the contact persons to be entered will need to be agreed locally but might
include the laboratory director, medical adviser and/or biosafety officer.
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The containment laboratory Biosafety Level 3
A. Front of card
ILLNESS SURVEILLANCE NOTICE
Card holders
picture
Name
TO THE EMPLOYEE
Keep this card in your possession. In case of unexplained
febrile illness, present the card to your physician and notify one
of the following in the order listed.
Work
Home
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Home
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B. Back of card
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TO THE PHYSICIAN
The holder of this card works in an area at
in which pathogenic viruses, rickettsia, bacteria, protozoa or
helminths are present. In the event of an unexplained febrile
illness, please call the employer for information on agents to
which this employee may have been exposed.
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Name of laboratory:
Address:
Tel:
Fig. 2. Suggested format for medical contact card
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5. The maximum containment laboratory

Biosafety Level 4
The maximum containment laboratory Biosafety Level 4 is designed for work with Risk Group 4
microorganisms. Before such a laboratory is constructed and put into operation, intensive consultations
should be held with institutions that have had experience of operating a similar facility. Operational
maximum containment laboratories Biosafety Level 4 should be under the control of national or other
appropriate health authorities.
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The features of a containment laboratory Biosafety Level 3 also apply to a maximum containment
laboratory Biosafety Level 4 with the addition of the following.
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1. Primary containment. An efficient primary containment system must be in place, consisting of one or
a combination of the following.
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Class III cabinet laboratory. Passage through a minimum of two doors prior to entering the rooms
containing the Class III biological safety cabinet (cabinet room) is required. In this laboratory
configuration the Class III biological safety cabinet provides the primary containment. A
personnel shower with inner and outer changing rooms is necessary. Supplies and materials that
are not brought into the cabinet room through the changing area are introduced through a
double-door autoclave or fumigation chamber. Once the outer door is securely closed, staff
inside the laboratory can open the inner door to retrieve the materials. The doors of the autoclave
or fumigation chamber are interlocked in such a way that the outer door cannot open unless the
autoclave has been operated through a sterilization cycle or the fumigation chamber has been
decontaminated (see Chapter 7)
.
Suit laboratory. A protective suit laboratory with self-contained breathing apparatus differs
significantly in design and facility requirements from a Biosafety Level 4 laboratory with Class III
biological safety cabinets. The rooms in the protective suit laboratory are arranged to direct
personnel through the changing and decontamination areas prior to entering areas where
infectious materials are manipulated. In this laboratory configuration, the suit area itself is
considered to constitute the primary containment. The suit area is designed and maintained to
provide personnel protection equivalent to that provided by Class III biological safety cabinets. A
suit decontamination shower must be provided for personnel leaving the containment laboratory
area. A separate personnel shower with inner and outer changing rooms is also provided.
Personnel who enter the suit area are required to don a one-piece, positively pressurized, HEPAfilter-supplied air suit. Air to the suit must be provided by a system that has a 100% redundant
capability with an independent source of air, for use in the event of an emergency. Entry into the
suit laboratory is through an airlock fitted with airtight doors. An appropriate warning system for
personnel working in the suit laboratory must be provided for use in the event of mechanical
system or air failure (see Chapter 7).
2. Controlled access. The maximum containment laboratory Biosafety Level 4 must be located in a
separate building or in a clearly delineated zone within a secure building. Entry and exit of personnel
and supplies must be through an airlock or pass-through system. On entering, personnel must put on
a complete change of clothing; before leaving, they should shower before putting on their street
clothing.
3. Controlled air system. Negative pressure must be maintained in the facility. Both supply and exhaust
air must be HEPA-filtered. There are significant differences in the ventilating systems of the Class III
cabinet laboratory and suit laboratory:
.
Class III cabinet laboratory. The supply air to the Class III biological safety cabinet(s) may be
drawn from within the room through a HEPA filter mounted on the cabinet or supplied directly
through the supply air system. Exhaust air from the Class III biological safety cabinet must pass
through two HEPA filters prior to release outdoors. The cabinet must be operated at negative
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The maximum containment laboratory Biosafety Level 4
Laboratory biosafety manual
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pressure to the surrounding laboratory at all times. A dedicated non-recirculating ventilating

system for the cabinet laboratory is required.
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Suit laboratory. Dedicated room air supply and exhaust systems are required. The supply and
exhaust components of the ventilating system are balanced to provide directional air flow within
the suit area from the area of least hazard to the area(s) of greatest potential hazard. Redundant
exhaust fans are required to ensure that the facility remains under negative pressure at all times.
The differential pressures within the suit laboratory and between the suit laboratory and adjacent
areas must be monitored. Air flow in the supply and exhaust components of the ventilating
system must be monitored and an appropriate system of controls must be used to prevent
pressurization of the suit laboratory. HEPA-filtered supply air must be provided to the suit area,
decontamination shower and decontamination airlocks or chambers. Exhaust air from the suit
laboratory must be passed through a series of two HEPA filters prior to release outdoors.
Alternatively, after double HEPA filtration, exhaust air may be recirculated but only within the suit
laboratory. Under no circumstances shall the exhaust air from the Biosafety Level 4 suit
laboratory be recirculated to other areas. Extreme caution must be exercised if recirculation of air
within the suit laboratory is elected. Consideration must be given to the types of research
conducted, equipment, chemicals and other materials used in the suit laboratory, as well as
animal species that may be involved in the research.
All HEPA filters need to be tested and certified annually. The HEPA filter housings are designed to
allow for in situ decontamination of the filter prior to removal. Alternatively, the filter can be removed
in a sealed, gas-tight primary container for subsequent decontamination and/or destruction by
incineration.
4. Decontamination of effluents. All effluents from the suit area, decontamination chamber,
decontamination shower, or Class III biological safety cabinet must be decontaminated before final
discharge. Heat treatment (autoclaving) is the preferred method. Effluents may also require
correction to a neutral pH prior to discharge. Water from the personnel shower and toilet may be
discharged directly to the sanitary sewer without treatment.
5. Sterilization of waste and materials. A double-door, pass-through autoclave must be available in the
laboratory area. Other methods of decontamination must be available for equipment and items that
cannot withstand steam sterilization.
6. Airlock entry ports for specimens, materials and animals must be provided.
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Because of the great complexity of the work in the Biosafety Level 4 laboratory, a separate detailed work
manual should be developed and tested in training exercises. In addition, an emergency programme
must be devised (see Chapter 13). In the preparation of this programme, active cooperation with national
and local health authorities should be established. Other emergency services, e.g. fire, police and
receiving hospitals, should also be involved.
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6. Laboratory animal facilities
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Those who use animals for experimental and diagnostic purposes have a moral obligation to take every
care to avoid causing them unnecessary pain or suffering. The animals must be provided with
comfortable, hygienic housing and adequate wholesome food and water. At the end of the experiment
they must be dealt with in a humane manner.
For security reasons, the animal house should be an independent, detached unit. If it adjoins a
laboratory, the design should provide for its isolation from the public parts of the laboratory should such
need arise, and for its decontamination and disinfestation.
Table 4. Animal facility containment levels: summary of practices and safety equipment
Containment
level
Laboratory practices and safety equipment
ABSL-1
Limited access, protective clothing and gloves.
ABSL-2
ABSL-1 practices plus: hazard warning signs. Class I or II BSCs for

activities that produce aerosols. Decontamination of waste and cages
before washing.
ABSL-3
ABSL-2 practices plus: Controlled access. BSCs and special protective

clothing for all activities.
ABSL-4
ABSL-3 plus: Strictly limited access. Clothing change before entering.

Class III BSCs or positive pressure suits. Shower on exit. Decontamination
of all wastes before removal from facility.
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ABSL, animal Biosafety Level; BSC, biological safety cabinet
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Animal facilities, like laboratories, may be designated primarily according to the risk group of the
microorganisms under investigation as Biosafety Level 1, 2, 3 or 4. Other factors should also be taken
into consideration. With respect to the agents, these include the normal route of transmission, the
volumes and concentrations to be used, the route of inoculation, and whether and by what route they
may be excreted. With respect to the animals, they include the nature of the animals, i.e. their
aggressiveness and tendency to bite and scratch, their natural ecto- and endoparasites, the zoonotic
diseases to which they are susceptible, and the possible dissemination of allergens.
As with laboratories, the requirements for design features, equipment and precautions increase in
stringency according to the biosafety level. These are described below and summarized in Table 4.
These guidelines are additive, so that each higher level incorporates the standards of the lower levels.
Animal facility Biosafety Level 1

This is suitable for the maintenance of most stock animals after quarantine (except nonhuman primates,
regarding which national authorities should be consulted) and for animals that are deliberately inoculated
with agents in Risk Group 1. Good microbiological technique is required. The animal facility director must
establish policies, procedures and protocols for all operations and for access to the vivarium. An
appropriate medical surveillance programme for the staff must be instituted. A safety or operations
manual must be prepared and adopted.
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Laboratory animal facilities

This is suitable for work with animals that are deliberately inoculated with microorganisms in Risk
Group 2. The following safety precautions apply.
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1. All the requirements for animal facilities Biosafety Level 1 must be met.
2. Biohazard warning signs (see Fig. 1) should be posted on doors and other appropriate places, and
should identify the infectious agent(s) in use.
3. The facility must be designed for easy cleaning and housekeeping.
4. Doors must open inwards and be self-closing.
5. Heating, ventilation and lighting must be adequate.
6. If mechanical ventilation is provided, the air flow must be inwards. Exhaust air is discharged to the
outside and should not be recirculated to any part of the building.
7. Access must be restricted to authorized persons.
8. No animals should be admitted other than those for experimental use.
9. There should be an arthropod and rodent control programme.
10. Windows, if present, must be secure, resistant to breakage and, if able to be opened, must be fitted
with arthropod-proof screens.
11. After use, work surfaces must be decontaminated with effective disinfectants (see Chapter 14).
12. Biological safety cabinets (Classes I or II) or isolator cages with dedicated air supplies and HEPAfiltered exhaust air must be provided for work that may involve the generation of aerosols.
13. An autoclave must be available on site or nearby.
14. Animal bedding materials must be removed in a manner that minimizes the generation of aerosols
and dust.
15. All waste materials and bedding must be decontaminated before disposal.
16. Use of sharp instruments should be restricted whenever possible. Sharps should always be collected
in puncture-proof containers fitted with covers and treated as infectious.
17. Material for autoclaving or incineration must be transported safely in closed containers.
18. Animal cages must be decontaminated after use.
19. Animal carcasses must be incinerated.
20. Protective clothing and equipment must be worn in the facility, and removed on leaving. Suitable
gloves should be available and be worn.
21. Hand-washing facilities must be provided. Staff must wash their hands before leaving the animal
facility.
22. All injuries, however minor, must be reported and recorded.
23. Eating, drinking and application of cosmetics must be forbidden in the facility.
24. All personnel must receive appropriate training.

This is suitable for work with animals that are deliberately inoculated with agents in Risk Group 3. All
systems, practices and procedures need to be reviewed and recertified annually.
1. All the requirements for animal facilities Biosafety Levels 1 and 2 must be met.
2. Access must be strictly controlled.
3. The facility must be separated from other laboratory and animal house areas by a room with two
doors, forming an anteroom.
4. Hand-washing facilities and showers must be provided in the anteroom.
5. There must be mechanical ventilation to ensure a continuous air flow through all the rooms. Exhaust
air must pass through HEPA filters before being discharged to the atmosphere without recirculation.
The system must be designed to prevent accidental reverse flow and positive pressurization in any
part of the animal house.
6. An autoclave must be available at a location convenient for the animal house where the biohazard is
contained. Infectious waste should be autoclaved before it is moved to other areas of the facility.
7. An incinerator should be readily available on site or alternative arrangements should be made with
the authorities concerned.
8. Animals infected with Risk Group 3 microorganisms must be housed in cages in isolators or rooms
with ventilation exhausts placed behind the cages.
9. Bedding should be as dust-free as possible.
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10. Laboratory protective clothing must be worn in the facility. This protective clothing must not be worn
outside the laboratory and it must be decontaminated before it is laundered.
11. Windows must be closed and sealed, and resistant to breakage.
12. Immunization of staff, as appropriate, should be offered.

Work in this facility will normally be linked with that in the maximum containment laboratory Biosafety
Level 4, and national and local rules and regulations must be harmonized to apply to both. If work is to be
done in a suit laboratory, additional practices and procedures must be used other than those described
here (see Chapter 5).
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Invertebrates
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1. All the requirements for animal facilities Biosafety Levels 1, 2 and 3 must be met.
2. Access must be strictly controlled; only staff designated by the director of the establishment should
have authority to enter.
3. Individuals must not work alone: the two-person rule must apply.
4. Personnel must have received the highest possible level of training as microbiologists and be familiar
with the hazards involved in their work and the necessary precautions.
5. Housing areas for animals infected with Risk Group 4 agents must maintain the criteria for
containment described and applied for maximum containment Biosafety Level 4 laboratories.
6. The facility must be entered by an airlock anteroom, the clean side of which must be separated from
the restricted side by changing and showering facilities.
7. The facility must be ventilated by a HEPA-filtered exhaust system designed to ensure a negative
pressure (inward directional air flow).
8. The ventilation system must be designed to prevent reverse flow and positive-pressurization.
9. A double-ended autoclave must be provided for delivery of materials, with the clean end in a room
outside the containment rooms.
10. Staff must remove street clothing when entering and put on special, protective clothing. After work
they must remove the protective clothing for autoclaving, and shower before leaving.
11. A pass-through airlock must be provided for delivery of materials.
12. All manipulations with animals infected with Risk Group 4 agents must take place under maximum
containment Biosafety Level 4 conditions.
13. All animals must be housed in isolators.
14. All bedding and waste must be autoclaved before removal from the facility.
15. There must be medical supervision of staff and immunization as appropriate.
The invertebrates that are used for experimental purposes in laboratories are usually the reservoirs or
vectors of pathogens or, as in the case of ecological and environmental investigations, may be
fortuitously infected with pathogens ingested with their food. They may include members of the following
phyla: Annelida, Aschelminthes, Arthropoda, Echinodermata, Mollusca, Platyhelminthes and Protozoa.
As with vertebrates, the animal facility biosafety level will be determined by the risk groups of the
agents under investigation or naturally present, but the following additional precautions are necessary
with certain arthropods, particularly with flying insects.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Separate rooms should be provided for infected and noninfected invertebrates.

The rooms should be capable of being sealed for fumigation.
Insecticide sprays should be readily available.
Chilling facilities should be provided to reduce, where necessary, the activity of invertebrates.
Access should be through an anteroom containing insect traps and with arthropod-proof screens on
the doors.
All exhaust ventilation ducts and openable windows should be fitted with arthropod-proof screens.
Waste traps on sinks and sluices should not be allowed to dry out.
All waste should be decontaminated by autoclaving, as some invertebrates are not killed by all
disinfectants.
A check should be kept on the numbers of larval and adult forms of flying, crawling and jumping
arthropods.
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10.
11.
12.
13.
Laboratory animal facilities
Containers for ticks and mites should stand in trays of oil.

Infected or potentially infected flying insects must be contained in double-netted cages.
Infected or potentially infected arthropods must be handled in biological safety cabinets or isolators.
Infected or potentially infected arthropods may be manipulated on cooling trays.
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For further information see references 1 and 6-9.
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PART II
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7. Biological safety cabinets
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Biological safety cabinets (BSCs) are designed to protect the operator, the laboratory environment and
work materials from exposure to infectious aerosols and splashes that may be generated when
manipulating materials containing infectious agents, such as primary cultures, stocks and diagnostic
specimens. Aerosol particles are created by any activity that imparts energy into a liquid or semiliquid
material, such as shaking, pouring, stirring or dropping liquid on to a surface or into another liquid.
Laboratory activities, such as streaking agar plates, inoculating cell culture flasks with a pipette, using a
multichannel pipette to dispense liquid suspensions of infectious agents into microculture plates,
homogenizing and vortexing infectious materials, and centrifugation of infectious liquids, or working with
animals, can generate infectious aerosols. Aerosol particles of less than 5 m in diameter and small
droplets of 5100 m in diameter are not visible to the naked eye. The laboratory worker is generally not
aware that such particles are being generated and may be inhaled or may cross-contaminate work
surface materials. BSCs, when properly used, have been shown to be highly effective in reducing
laboratory-acquired infections and cross-contaminations of cultures due to aerosol exposures. Each BSC
also protects the environment.
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Table 5. Selection of a biological safety cabinet (BSC), by type of protection needed

BSC selection
Personnel protection, microorganisms in Risk

Groups 13
Class I, Class II, Class III

Class III
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Group 4, glove box laboratory
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Type of protection
Class I, Class II
Product protection
Class II, Class III only if laminar flow included
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Class IIB1, Class IIA2 vented to the outside
Volatile radionuclide/chemical protection
Class I, Class IIB2, Class III
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Volatile radionuclide/chemical protection, minute

amounts
Over the years the basic design of BSCs has undergone several design modifications. A major
change was the addition of a high-efficiency particulate air (HEPA) filter to the exhaust system. The
HEPA filter traps 99.97% of particles of 0.3 m in diameter and 99.99% of particles of greater or smaller
size. This enables the HEPA filter to effectively trap all known infectious agents and ensure that only
microbe-free exhaust air is discharged from the cabinet. A second design modification was to direct
HEPA-filtered air over the work surface, providing protection of work surface materials from
contamination. This feature is often referred to as product protection. These basic design concepts have
led to the evolution of three classes of BSCs. The type of protection provided by each is set out in
Table 5.
Note. Horizontal and vertical outflow cabinets (clean-air work stations) are not biological safety cabinets
and should not be used as such.
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Class I biological safety cabinet
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Fig. 3 provides a schematic diagram of a Class I BSC. Room air is drawn in through the front opening at
a minimum velocity of 0.38 m/s, it passes over the work surface and is discharged from the cabinet
through the exhaust duct. The directional flow of air whisks aerosol particles that may be generated on
the work surface away from the laboratory worker and into the exhaust duct. The front opening allows the
operators arms to reach the work surface inside the cabinet while he or she observes the work surface
through a glass window. The window can also be fully raised to provide access to the work surface for
cleaning or other purposes.
The air from the cabinet is exhausted through a HEPA filter: (a) into the laboratory and then to the
outside of the building through the building exhaust; (b) to the outside through the building exhaust; or (c)
directly to the outside. The HEPA filter may be located in the exhaust plenum of the BSC or in the
building exhaust. Some Class I BSCs are equipped with an integral exhaust fan, whereas others rely on
the exhaust fan in the building exhaust system.
The Class I BSC was the first recognized BSC and, because of its simple design, it is still in wide use
throughout the world. It has the advantage of providing personnel and environmental protection and can
also be used for work with radionuclides and volatile toxic chemicals. Because unsterilized room air is
drawn over the work surface through the front opening, it is not considered to provide consistently reliable
product protection.
Fig. 3. Schematic diagram of a Class I biological safety cabinet. A: front opening, B: sash, C: exhaust
HEPA filter, D: exhaust plenum.
Class II biological safety cabinets

As the use of cell and tissue cultures for the propagation of viruses and other purposes grew, it was no
longer considered satisfactory for unsterilized room air to pass over the work surface. The Class II BSC
was designed not only to provide personnel protection but also to protect work surface materials from
contaminated room air. Class II BSCs, of which there are four types (A1, A2, B1 and B2), differ from
Class I BSCs by allowing only HEPA-filtered (sterile) supply air to flow over the work surface. The Class II
BSC can be used for working with infectious agents in Risk Groups 2 and 3. Class II BSCs can be used
for working with infectious agents in Risk Group 4 when positive pressure suits are used.
Class II type A1 biological safety cabinet

The Class II type A BSC is shown in Fig. 4. An internal fan draws room air (supply air) into the cabinet
through the front opening and into the front intake grill. The inflow velocity of this air should be at least
0.38 m/s at the face of the front opening. The supply air then passes through a supply HEPA filter before
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Biological safety cabinets
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flowing downwards over the work surface. As the air flows downwards it splits about 618 cm from the
work surface, one half of the downwards flowing air passing through the front exhaust grill, and the other
half passing through the rear exhaust grill. Any aerosol particles generated at the work surface are
immediately captured in this downward air flow and passed through the front or rear exhaust grills,
thereby providing the highest level of product protection. The air is then discharged through the rear
plenum into the space between the supply and exhaust filters located at the top of the cabinet. Owing to
the relative size of these filters, about 70% of the air recirculates through the supply HEPA filter back into
the work zone; the remaining 30% passes through the exhaust filter into the room or to the outside.
Air from the Class IIA1 BSC exhaust can be recirculated to the room or discharged to the outside of
the building through a thimble connection to a dedicated duct or through the building exhaust system.
Recirculating the exhaust air to the room has the advantage of lowering building fuel costs because
heated and/or cooled air is not being passed to the outside environment. A connection to a ducted
exhaust system also allows some BSCs to be used for work with volatile radionuclides and volatile toxic
chemicals (Table 5).
Fig. 4. Schematic representation of a Class IIA1 biological safety cabinet. A: front opening, B: sash,
C: exhaust HEPA filter, D: rear plenum, E: supply HEPA filter, F: blower.
Class II type A2 vented to the outside, B1 and B2 biological safety cabinets

Class IIA2 vented to the outside, IIB1 (Fig. 5) and IIB2 BSCs are variations of the type IIA1, and their
characteristics, along with those of Class I and Class III BSCs, are indicated in Table 6. Each variation
allows the BSC to be used for specialized purposes (see Table 5). These BSCs differ from one another in
several aspects: the air intake velocity through the front opening; the amount of air recirculated over the
work surface and exhausted from the cabinet; the exhaust system, which determines whether air from the
cabinet is exhausted to the room, or to the outside, through a dedicated exhaust system or through the
building exhaust; and the pressure arrangements (whether cabinets have biologically contaminated ducts
and plenums under negative pressure, or have biological contaminated ducts and plenums surrounded
by negative-pressure ducts and plenums).
Complete descriptions of the various Class IIA and IIB BSCs can be obtained from references 10 and 11,
and from manufacturers brochures.
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Fig. 5. Schematic diagram of a Class IIB1 biological safety cabinet. A: front opening, B: sash, C:
exhaust HEPA filter, D: supply HEPA filter, E: negative pressure exhaust plenum, F: blower, G: HEPA
filter for supply air. Connection of cabinet exhaust to building exhaust air system is required.
0.36
Class IIA1
0.380.51
Recirculated
Exhausted
100
Hard duct
70
30
Exhaust to room or thimble

connection
70
30
Exhaust to room or thimble

connection
0.51
30
70
Hard duct
0.51
100
Hard duct
100
Hard duct
Class IIB2
Exhaust system
0.51
Class IIB1
Class III
% of air flow
Class IIA2
vented to the
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Face velocity
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Table 6. Differences between Class I, II and III biological safety cabinets (BSCs)
NA
All biologically contaminated ducts are under negative pressure, or are surrounded by negative pressure ducts and plenums.
NA, not applicable.
Class III biological safety cabinet

This type (Fig. 6) provides the highest level of personnel protection and is used for Risk Group 4 agents.
All penetrations are sealed gas tight. Supply air is HEPA-filtered and exhaust air passes through two
HEPA filters. Air flow is maintained by a dedicated exhaust system exterior to the cabinet, which keeps
the cabinet interior under negative pressure (about 124.5 Pa). Access to the work surface is by means of
heavy duty rubber gloves, which are attached to ports in the cabinet. The Class III BSC should have an
attached pass-through box that is sterilizable and is equipped with HEPA-filtered exhaust. The Class III
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cabinet may be connected to a double-door autoclave used to decontaminate all materials entering or
exiting the cabinet. Several glove boxes can be joined together to extend the work surface. Class III
BSCs are suitable for work in Biosafety Level 3 and 4 laboratories.
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Fig. 6. Schematic representation of a Class III biological safety cabinet (glove box). A: glove ports
for arm-length gloves, B: sash, C: double exhaust HEPA filters, D: supply HEPA filter, E: double-ended
autoclave or pass-through box, F: chemical dunk tank. Connection of cabinet exhaust to an independent
building exhaust air system is required.
Biological safety cabinet air connections
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A thimble or canopy hood is designed for use with Class IIA1 and IIA2 vented to the outside BSCs.
The thimble fits over the cabinet exhaust housing, sucking the cabinet exhaust air into the building
exhaust ducts. A small opening, usually 2.5 cm in diameter, is maintained between the thimble and the
cabinet exhaust housing. This small opening enables room air to be sucked into the building exhaust
system as well. The building exhaust capacity must be sufficient to capture both room air and the cabinet
exhaust. The thimble must be removable or be designed to allow for operational testing of the cabinet.
Generally, the performance of a thimble-connected BSC is not affected much by fluctuations in the air
flow of the building
Class IIB1 and IIB2 BSCs are hard-ducted, i.e. firmly connected without any openings, to the building
exhaust system or, preferably, to a dedicated exhaust duct system. The building exhaust system must
precisely matched to the air flow requirements specified by the manufacturer in both volume and static
pressure. Certification of hard-duct connected BSCs is more time-consuming than that for BSCs that
recirculate air to the room or which are thimble-connected.
Selection of a biological safety cabinet

A BSC should be selected primarily in accordance with the type of protection needed: product protection;
personnel protection against Risk Group 14 microorganisms; personnel protection against exposure to
radionuclides and volatile toxic chemicals; or a combination of these. Table 5 shows which BSCs are
recommended for each type of protection.
Volatile or toxic chemicals should not be used in BSCs that recirculate exhaust air to the room, i.e.
Class I that are not ducted to building exhaust systems, or Class IIA1 or Class IIA2 cabinets. Class IIB1
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cabinets are acceptable for work with minute amounts of volatile chemicals and radionuclids. A
Class IIB2 cabinet, also called a total exhaust cabinet, is necessary when significant amounts of
radionuclides and volatile chemicals are expected to be used.
Using biological safety cabinets in the laboratory

Location
The velocity of air flowing through the front opening into a BSC is about 0.45 m/s. At this velocity the
integrity of the directional air inflow is fragile and can be easily disrupted by air currents generated by
people walking close to the BSC, open windows, air supply registers, and opening and shutting doors.
Ideally, BSCs should be situated in a location remote from traffic and potentially disturbing air currents.
Whenever possible a 30-cm clearance should be provided behind and on each side of the cabinet to
allow easy access for maintenance. A clearance of 3035 cm above the cabinet may be required to
provide for accurate air velocity measurement across the exhaust filter and for exhaust filter changes.
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If BSCs are not used properly, their protective benefits may be greatly diminished. Operators need to be
careful to maintain the integrity of the front opening air inflow when moving their arms into and out of
cabinets. Arms should be moved in and out slowly, perpendicular to the front opening. Manipulations of
materials within BSCs should be delayed for about 1 min after placing hands and arms inside to allow the
cabinet to adjust and to air sweep the surface of the hands and arms. The number of movements
across the front opening should also be minimized by placing all necessary items into the cabinet before
beginning manipulations.
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The front intake grill of Class II BSCs must not be blocked with paper, equipment or other items.
Materials to be placed inside the cabinet should be surface-decontaminated with 70% alcohol. Work may
be performed on disinfectant-soaked absorbent towels to capture splatters and splashes. All materials
should be placed as far back in the cabinet, towards the rear edge of the work surface, as practical
without blocking the rear grill. Aerosol-generating equipment (e.g. mixers, centrifuges, etc.) should be
placed towards the rear of the cabinet. Bulky items, such as biohazard bags, discard pipette trays and
suction collection flasks should be placed to one side of the interior of the cabinet. Active work should
flow from clean to contaminated areas across the work surface.
The autoclavable biohazard collection bag and pipette collection tray should not be placed outside the
cabinet. The frequent in-and-out movement needed to use these containers is disruptive to the integrity of
the cabinets air barrier, and can compromise both personnel and product protection.
Operation and maintenance

Most BSCs are designed to permit operation 24 h/day, and investigators find that continuous operation
helps to control the levels of dust and particulate materials in the laboratory. Class IIA1 and IIA2 BSCs
exhausting to the room or connected by thimble connections to dedicated exhaust ducts can be turned off
when not in use. Other types such as IIB1 and IIB2 BSCs, which have hard-duct installations, must have
air flow through them at all times to help maintain room air balance. Cabinets should be turned on at least
5 min before beginning work and after completion of work to allow the cabinet to purge, i.e. to allow time
for contaminated air to be removed from the cabinet environment.
All repairs made on BSCs should be made by a qualified technician. Any malfunction in the operation
of the BSC should be reported and repaired before the BSC is used again.
Ultraviolet lights
Ultraviolet lights are not required in BSCs. If they are used, they must be cleaned weekly to remove any
dust and dirt that may block the germicidal effectiveness of the light. Ultraviolet light intensity should be
checked when the cabinet is recertified to ensure that light emission is appropriate. Ultraviolet lights must
be turned off while the room is occupied, to protect eyes and skin from inadvertent exposure.
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Open flames
Open flames should be avoided in the near microbe-free environment created inside the BSC. They
disrupt the air flow patterns and can be dangerous when volatile, flammable substances are also used.
To sterilize bacteriological loops, microburners or electric furnaces are available and are preferable to
open flames.
Spills
A copy of the laboratorys protocol for handling spills should be posted, read and understood by everyone
who uses the laboratory. When a spill of biohazardous material occurs within a BSC, cleanup should
begin immediately, while the cabinet continues to operate. An effective disinfectant should be used and
applied in a manner that minimizes the generation of aerosols. All materials that come into contact with
the spilled agent should be disinfected and/or autoclaved.
Annual certification
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The functional operation and integrity of each BSC should be certified to national or international
performance standards at the time of installation and regularly thereafter by qualified technicians,
according to the manufacturers instructions. Evaluation of the effectiveness of cabinet containment
should include tests for cabinet integrity, HEPA filter leaks, downflow velocity profile, face velocity,
negative pressure/ventilation rate, air flow smoke pattern, and alarms and interlocks. Optional tests for
electrical leaks, lighting intensity, ultraviolet light intensity, noise level and vibration may also be
conducted. Special training, skills and equipment are required to perform these tests and it is highly
recommended that they are undertaken by a qualified professional.
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Cleaning and disinfection
Decontamination
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All items within BSCs, including equipment, should be surface-decontaminated and removed from the
cabinet when work is completed, since residual culture media may provide an opportunity for microbial
growth.
The interior surfaces of BSCs should be decontaminated before and after each use. The work
surfaces and interior walls should be wiped with a disinfectant that will kill any microorganisms that might
be found inside the cabinet. At the end of the work day, the final surface decontamination should include
a wipe-down of the work surface, the sides, back and interior of the glass. A solution of bleach or 70%
alcohol should be used where effective for target organisms. A second wiping with sterile water is needed
when a corrosive disinfectant, such as bleach, is used.
It is recommended that the cabinet be left running. If not, it should be run for 5 min in order to purge
the atmosphere inside before it is switched off.
BSCs must be decontaminated before filter changes and before being moved. The most common
decontamination method is by fumigation with formaldehyde gas. BSC decontamination should be
performed by a qualified professional.
Personal protective equipment

Personal protective clothing should be worn whenever using a BSC. Laboratory coats are acceptable for
work being performed at Biosafety Levels 1 and 2. A solid front, back-closing laboratory gown provides
better protection and should be used at Biosafety Levels 3 and 4 (except for suit laboratories). Gloves
should be pulled over the wrists of the gown rather than worn inside. Elasticized sleeves can be worn to
protect the investigators wrists. Masks and safety glasses may be required for some procedures.
Alarms
BSCs can be equipped with one of two kinds of alarm. Sash alarms are found only on cabinets with
sliding sashes. The alarm signifies that the operator has moved the sash to an improper position.
Corrective action for this type of alarm is returning the sash to the proper position. Air-flow alarms
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indicate a disruption in the cabinets normal air-flow pattern. This represents an immediate danger to the
operator or product. When an air-flow alarm sounds, work should cease immediately and the laboratory
supervisor should be notified. Manufacturers instruction manuals should provide further details. Training
in the use of BSCs should cover this aspect.
Supplementary information
Selecting the correct type of BSC, installing it, using it properly and annually certifying its operation are
complex processes. It is highly recommended that they proceed under the supervision of a well-trained
and experienced biosafety professional. The professional should be highly familiar with the relevant
literature listed in the References section, and should have been trained on all aspects of BSCs.
Operators should receive formal training in the operation and use of BSCs.
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For further information see references 1 and 10-19, and Chapter 10.
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8. Equipment-related hazards
Certain items of equipment may create microbiological hazards when they are used. Other items are
specifically designed to prevent or reduce biological hazards (see Chapter 9).
Equipment that may create a hazard
Table 7. Equipment and operations that may create hazards
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Table 7 lists equipment and operations that may create hazards and suggests how such hazards may be
eliminated or reduced.
Hazard
How to eliminate or reduce the hazard
Hypodermic
needles
Accidental inoculation,
aerosol or spillage (20)
Do not recap or clip needles.
Use a needle-locking type of syringe to prevent

separation of needle and syringe, or use a disposable
type where the needle is an integral part of the
syringe unit.
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Equipment
Use good laboratory techniques, e.g.:

fill the syringe carefully to minimize air bubbles
and frothing of inoculum
avoid using syringes to mix infectious liquids; if

used, ensure that the tip of the needle is held
under the surface of the fluid in the vessel and
avoid excessive force
wrap the needle and stopper in a cotton pledget

moistened with an appropriate disinfectant before
withdrawing the needle from a rubber-stoppered
bottle
expel excess liquid and air bubbles from the

syringe vertically into a cotton pledget moistened
with an appropriate disinfectant or into a small
bottle containing cotton.
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Use a biological safety cabinet for all operations with

infectious material.
Restrain animals while they are being inoculated. Use

blunt needles or cannulas for intranasal or oral
inoculation. Use a biological safety cabinet.
Autoclave after use and ensure proper disposal. If a

disposable needle and syringe unit is used, do not
disassemble prior to autoclaving.
Centrifuges
Aerosols, splashing and

tube breakage
Use sealable buckets (safety cups) or sealed rotors.

Open buckets or rotors after aerosols have settled
(30 min) or in a biological safety cabinet.
Ultra-
Install a HEPA filter between centrifuge and vacuum
37
Equipment
Hazard
centrifuges
tube breakage
How to eliminate or reduce the hazard

pump.
Maintain a logbook of operating hours for each rotor

and a preventive maintenance programme to reduce
risk of mechanical failure.
Load and unload buckets or rotors in a biological

safety cabinet.
Explosion, dispersing
infectious materials
Ensure integrity of wire capsule around catalyst.
Desiccators
Implosion, dispersing
glass fragments and
infectious materials
Place in a stout wire cage.
Homogenizer,
tissue grinders
Aerosols, leakage and

container breakage
Operate and open equipment in a biological safety

cabinet.
Use specially designed models that prevent leakage

from rotor bearings and O-ring gaskets, or use a
stomacher.
Before opening the blender bowl, wait 30 min to allow

the aerosol cloud to settle. Refrigerate to condense
aerosols.
If manual tissue grinders are used, hold tube in a wad

of absorbent material.

spillage
Freeze-dryers
(lyophilizers)
Aerosols and direct

contact contamination
Growth of
microorganisms. Sodium
azide forms explosive
compounds with some
metals.
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Operate and open equipment in a biological safety

cabinet or sealed unit.
Ensure insulation to protect against subharmonics.
Wear gloves for protection against high-frequency

plus detergent action on skin.
Operate in a biological safety cabinet or specially

designed primary containment.
Use heavy-duty screw-capped culture flasks, fitted

with filter-protected outlets, if necessary, and well
secured.
Use O-ring connectors to seal the unit throughout.
Use air filters to protect vacuum lines.
Use a satisfactory method of decontamination, e.g.

chemical.
Provide an all-metal moisture trap and a vapour

condenser.
Carefully inspect all glass vacuum vessels for surface

scratches. Use only glassware designed for vacuum
work.
Ensure regular cleaning and disinfection.
Do not use sodium azide for preventing growth of

organisms.
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Culture
stirrers,
shakers,
agitators
Water baths
and Warburg
baths
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Aerosols, impaired
hearing, dermatitis
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Sonicators,
ultrasonic
cleaners
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Anaerobic jars
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9. Equipment designed
to reduce biological hazards
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As aerosols are important sources of infection, care should be taken to reduce the extent of their
formation and dispersion. Hazardous aerosols can be generated by many laboratory operations, e.g.
blending, mixing, grinding, shaking, stirring, sonicating and centrifuging of infectious materials. Even
when safe equipment is used, it is best to carry out these operations in an approved biological safety
cabinet whenever possible. Biological safety cabinets and their use and testing are discussed in
Chapter 7. The use of safety equipment is no assurance of protection unless the operator is trained and
uses proper techniques. Equipment should be tested regularly to ensure its continued safe performance.
Table 8 provides a checklist of safety equipment designed to eliminate or reduce certain hazards and
briefly outlines the safety features. Further details of much of this equipment are given in subsequent
pages. Additional information on its proper use is provided in Chapter 10.
Hazard corrected
Safety features
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Equipment
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Table 8. Biosafety equipment
Minimum inward air flow (face

velocity) at work access opening.
Adequate filtration of exhaust air
Minimum inward air flow (face

velocity) at work access opening.
Adequate filtration of exhaust air
Aerosol and spatter
Maximum containment
Class I
Aerosol and spatter
Class II
Aerosol and spatter
Class III
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Biological safety
cabinet
Aerosol and spatter
Maximum containment
Spatter shield
Spatter of chemicals
Forms screen between operator

and work
Hazards from pipetting by mouth,

e.g. ingestion of pathogens,
inhalation of aerosols produced
by mouth suction on pipette,
blowing out of liquid or dripping
from pipette, contamination of
suction end of pipette
Ease of use
Control contamination of suction

end of pipette, protecting pipetting
aid, user and vacuum line
Can be sterilized
Control leakage from pipette tip
Spatter from transfer loops
Shielded in open-ended glass or

ceramic tube, heated by gas or
electricity
Disposable, no heating necessary
Leakproof construction with lid or

cover
Durable
Autoclavable
Pipetting aids
Loop
microincinerators,
disposable loops
Leakproof vessels for

collection and
transport of infectious
materials for
sterilization within a
in
Negative pressure
flexible-film isolator
Aerosols, spillage and leakage
39
Equipment
Hazard corrected
Safety features
Sharps disposal
containers
Accidental puncture wounds
Autoclavable
Robust, puncture proof
Transport containers
between laboratories,
institutions
Accidental escape of
microorganisms
Robust
Water-tight primary and secondary

containers to contain spills
Absorbent material to contain spills
facility
Infectious material (made safe for

disposal or reuse)
Approved design
Effective heat sterilization
Screw-capped bottles
Aerosols and spillage
Effective containment
Vacuum line
protection
Contamination of laboratory
vacuum system with aerosols and
overflow fluids
Cartridge-type filter prevents

passage of aerosols (particle size
0.45 m)
Overflow flask contains appropriate

disinfectant. Rubber bulb may be
used to close off vacuum
automatically when storage flask is
full
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Autoclaves, manual or
automatic
Entire unit autoclavable
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Negative-pressure flexible-film isolators
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The negative-pressure flexible-film isolator is a self-contained primary containment device that provides
maximum protection against hazardous biological materials. It may be mounted on a mobile stand. The
workspace is totally enclosed in a transparent polyvinylchloride (PVC) envelope suspended from a steel
framework. The isolator is maintained at an internal pressure lower than atmospheric pressure. Inlet air is
passed through one HEPA filter and outlet air is passed through two HEPA filters, thus obviating the need
to duct exhaust air outside the building. The isolator may be fitted with an incubator, microscope and
other laboratory equipment, such as centrifuges, animal cages, heat blocks, etc. Material is introduced
and removed from the isolator through supply and sample ports without compromising microbiological
security. Manipulations are performed using gloved sleeves incorporating disposable gloves. A
manometer is installed to monitor envelope pressure.
Flexible-film isolators are used for high-risk organisms (Risk Groups 3 or 4) where it is not feasible to
install or maintain conventional biological safety cabinets. They may be transported for use in field work.
Pipetting aids
A pipetting aid must always be used for pipetting procedures. Mouth pipetting must be strictly forbidden.
The importance of pipetting aids cannot be overemphasized. The most common hazards associated
with pipetting procedures are the result of mouth suction. Oral aspiration and ingestion of hazardous
materials have been responsible for many laboratory-associated infections and accidents.
Pathogens can also be transferred to the mouth if a contaminated finger is placed on the suction end
of a pipette. A lesser known hazard of mouth pipetting is the inhalation of aerosols caused by suction.
The cotton plug is not an efficient microbial filter at negative or positive pressure, and particles may be
sucked through it. Violent suction may be applied when the plug is tightly packed, resulting in the
aspiration of plug, aerosol and even liquid. The ingestion of pathogens is prevented by the use of
pipetting aids.
Aerosols can also be generated when a liquid is dropped from a pipette on to a work surface, when
cultures are mixed by alternate sucking and blowing, and when the last drop is blown out of a pipette.
40
Equipment designed to reduce biological hazards
The inhalation of aerosols unavoidably generated during pipetting operations can be prevented by
working in a biological safety cabinet.
Pipetting aids should be selected with care. Their design and use should not create an additional
infectious hazard and they should be easy to sterilize and clean. Plugged (aerosol-resistant) pipette tips
should be used when manipulating microorganisms and cell cultures.
Pipettes with cracked or chipped suction ends should not be used as they damage the seating seals
of pipetting aids and so create a hazard.
Homogenizers, shakers, blenders and sonicators
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Domestic (kitchen) homogenizers are not sealed and release aerosols. Only equipment designed for
laboratory use should be used. Their construction minimizes or prevents such release. Stomachers,
which are now available for use with large and small volumes, may also produce aerosols.
Homogenizers used for Risk Group 3 microorganisms should always be loaded and reopened in
biological safety cabinets.
Sonicators may release aerosols. They should be operated in biological safety cabinets or covered
with shields during use. The shields and outsides of sonicators should be decontaminated after use.
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Disposable transfer loops
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The advantage of disposable transfer loops is that they do not have to be sterilized and can therefore be
used in biological safety cabinets where Bunsen burners and microincinerators would disturb the air flow.
These loops should be placed in disinfectant after use and discarded as contaminated waste (see section
on Contaminated (infectious) materials for disposal in Chapter 3).
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Gas- and electrically-heated microincinerators have borosilicate glass or ceramic shields that minimize
the spatter and dispersal of infected material when transfer loops are sterilized. However,
microincinerators disturb the air flow and should therefore not be used in biological safety cabinets.
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Personal protective clothing and equipment
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Personal protective clothing and equipment may act as a barrier to minimize the risk of exposure to
aerosols, splashes and accidental inoculation. The clothing and equipment selected is dependent on the
nature of the work performed. Protective clothing should be worn when working in the laboratory. Before
leaving the laboratory, protective clothing should be removed, and hands should be washed. Table 9
summarizes some personal protective equipment used in laboratories and the protection afforded.
Table 9. Personal protective equipment
Equipment
Hazard corrected
Safety features
Laboratory coats,
gowns, coveralls
Contamination of clothing
Back opening
Cover street clothing
Plastic aprons
Contamination of clothing
Waterproof
Footwear
Impact and splash
Closed toe
Goggles or safety
spectacles
Impact and splash
Impact-resistant lenses (must be optically

correct or worn over corrective eye glasses)
Side shields
Face shields
Impact and splash
Shield entire face

Easily removable in case of accident
Respirators
Inhalation of aerosols
Designs available include hoods, full-face or
41
Equipment
Hazard corrected
Safety features
half face masks
Gloves
Accidental direct contact

Accidental punctures or cuts
Disposable latex or vinyl

Hand protection
Mesh
Laboratory coats, gowns, coveralls, aprons

Laboratory coats should preferably be fully buttoned. However, long-sleeved, back-opening gowns or
coveralls give better protection than laboratory coats and are preferred in microbiology laboratories and
when working in the biological safety cabinet. Aprons should be worn over laboratory coats or gowns
where necessary to give further protection against spillage of chemicals or biological materials such as
blood or culture fluids.
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Goggles, safety spectacles, face shields
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The choice of equipment to protect the eyes and face from splashes and impacting objects will depend
on the activity performed. Prescription or plain eye glasses can be manufactured with special frames that
allow lenses to be placed in frame from the front, using shatterproof material either curved or fitted with
side shields (safety glasses). Goggles should be worn over normal prescription eye glasses and contact
lenses, which do not provide protection against biological hazards. Face shields (visors) are made of
shatterproof plastic, fit over the face and are held in place by head straps or caps.
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Respirators
Gloves
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Respiratory protection may be used when carrying out high-hazard procedures (e.g. cleaning up a spill of
infectious material). The choice between mask and respirator, and type of respirator will depend on the
type of hazard. Respirators are available with interchangeable filters for protection against gases,
vapours, particulates and microorganisms. Note that no filter other than a HEPA filter will provide
protection against microorganisms, and it is imperative that the filter be fitted in the correct type of
respirator. To achieve optimal protection, respirators should be individually fitted to the operators face
and tested. Fully self-contained respirators with an integral air supply provide full protection. Advice
should be sought from a suitably qualified person, e.g. an occupational hygienist, for selection of the
correct respirator.
Contamination of hands may occur when laboratory procedures are performed. Hands are also
vulnerable to sharps injuries. Disposable latex or vinyl surgical-type gloves are used widely for general
laboratory work, and for handling infectious agents and blood and body fluids. Reusable gloves may also
be used but attention must be given to their correct washing, removal, cleaning and disinfection.
Gloves should be removed and hands thoroughly washed after handling infectious materials, working
in a biological safety cabinet and before leaving the laboratory. Used disposable gloves should be
discarded with infected laboratory wastes.
Allergic reactions such as dermatitis and immediate hypersensitivity have been reported in laboratory
and other workers wearing latex gloves, particularly those with powder. Alternatives such as powder-free
latex or vinyl gloves should be used if problems occur.
Stainless steel mesh gloves should be worn when there is a potential exposure to sharp instruments
e.g. during postmortem examinations. However, such gloves protect against slicing motion but do not
protect against needle injury.
For further information see references 12, 20 and 21.
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PART III
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Good microbiological technique
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10. Safe laboratory techniques
Human error, poor laboratory techniques and misuse of equipment cause the majority of laboratory
accidents, injuries and work-related infections. This chapter provides a compendium of technical methods
that are designed to avoid or minimize the most commonly reported problems of this nature.
Safe handling of specimens in the laboratory
es
Improper collection, internal transport and receipt of specimens in the laboratory carry a risk of infection
to the personnel involved.
Specimen containers
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Specimen containers may be of glass or preferably plastic. They should be robust and should not leak
when the cap or stopper is correctly applied. No material should remain on the outside of the container.
Containers should be correctly labelled to facilitate identification. Specimen request or specification forms
should not be wrapped around the containers but placed in separate, preferably waterproof envelopes.
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Transport of specimens within the facility
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Receipt of specimens
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To avoid accidental leakage or spillage, secondary containers, such as boxes, should be used, fitted with
racks so that the specimen containers remain upright. The secondary containers may be of metal or
plastic, should be autoclavable or resistant to the action of chemical disinfectants, and the seal should
preferably have a gasket. They should be regularly decontaminated. Transport of specimens to the
facility from distant locations is described in Chapter 12.
Opening packages
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Laboratories that receive large numbers of specimens should designate a particular room or area for this
purpose.
Personnel who receive and unpack specimens should be aware of the potential health hazards involved,
and should be trained to adopt universal precautions (3), particularly when dealing with broken or leaking
containers. Specimens should be unpacked in a biological safety cabinet. Disinfectants should be
available.
Use of pipettes and pipetting aids

1.
2.
3.
4.
5.
6.
7.
A pipetting aid should always be used. Pipetting by mouth must be prohibited.

All pipettes should have cotton plugs to reduce contamination of pipetting devices.
Air should never be blown through a liquid containing infectious agents.
Infectious materials should not be mixed by alternate suction and expulsion through a pipette.
Liquids should not be forcibly expelled from pipettes.
Mark-to-mark pipettes are preferable to other types as they do not require expulsion of the last drop.
Contaminated pipettes should be completely submerged in a suitable disinfectant contained in an
unbreakable container. They should be left in the disinfectant for 18-24 h before disposal.
8. A discard container for pipettes should be placed within the biological safety cabinet, not outside it.
45
9. Syringes fitted with hypodermic needles must not be used for pipetting. Blunt cannulas should be
used instead of needles. There are devices for opening septum-capped bottles that allow pipettes to
be used and avoid the use of hypodermic needles and syringes.
10. To avoid dispersion of infectious material accidentally dropped from a pipette, a disinfectant-soaked
cloth or absorbent paper should be placed on the working surface; this should be autoclaved or
discarded as infectious waste after use.
Avoiding the dispersal of infectious materials
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1. In order to avoid the premature shedding of their loads, microbiological transfer loops should have a
diameter of 23 mm and be completely closed. The shanks should be not more than 6 cm in length to
minimize vibration.
2. The risk of spatter of infectious material in an open Bunsen burner flame should be avoided by using
an enclosed electric microincinerator to sterilize transfer loops. Disposable transfer loops, which do
not need to be resterilized, are preferable.
3. Catalase tests should not be performed on slides to avoid bubbling and dispersal of aerosols. The
tube, capillary tube or cover-glass methods should be used instead.
4. Discarded specimens and cultures for autoclaving and/or disposal should be placed in leakproof
containers, e.g. laboratory discard bags.
5. Working areas must be decontaminated with a suitable disinfectant at the end of each work period.
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For further information see reference 12.
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Use of biological safety cabinets
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1. The use and limitations of biological safety cabinets should be explained to all potential users (see
Chapter 7), with reference to national standards and relevant literature. Written protocols or safety or
operations manuals should be issued to staff. In particular, it must be made clear that the cabinet will
not protect the operator from spillage, breakage or poor technique.
2. The cabinet must not be used unless it is working properly.
3. The glass viewing panel must not be opened when the cabinet is in use.
4. Apparatus and materials in the cabinet must be kept to a minimum. Air circulation at the rear plenum
must not be blocked. Materials should be surface-decontaminated before placing them inside the
working area of the cabinet.
5. Bunsen burners must not be used in the cabinet. The heat produced will distort the air flow and may
damage the filters. An electric microincinerator is permissible but sterile disposable transfer loops are
better.
6. All work must be carried out in the middle or rear part of the working surface and be visible through
the viewing panel.
7. Traffic behind the operator should be minimized.
8. The operator should not disturb the air flow by repeated removal and reintroduction of his or her
arms.
9. The front air grill must not be blocked with notes, pipettes or other materials, as this will disrupt the air
flow causing potential contamination of the material and exposure of the operator.
10. The surface of the biological safety cabinet should be wiped using an appropriate disinfectant after
work is completed and at the end of the day.
11. The cabinet fan should be run for at least 5 min before beginning work and after completion of work
in the cabinet.
For further information about biological safety cabinets see Chapter 7.
Avoiding ingestion of infectious materials and contact with skin and eyes
1. Large particles and droplets (> 5 m in diameter) released during microbiological manipulations settle
rapidly on bench surfaces and on the hands of the operator. Disposable gloves should be worn.
Laboratory workers should avoid touching their mouth, eyes and face.
2. Food and drink must not be consumed or stored in the laboratory.
3. There should be no gum-chewing in the laboratory.
46
Safe laboratory techniques
4. Cosmetics should not be applied in the laboratory.

5. The face, eyes and mouth should be shielded or otherwise protected during any operation that may
result in the splashing of potentially infectious materials.
Avoiding injection of infectious materials
es
1. Accidental inoculation with broken or chipped glassware can be avoided through careful practices
and procedures. Glassware should be replaced with plastic ware whenever possible.
2. Injections may result from accidents with hypodermic needles (needle-sticks), glass Pasteur pipettes
and broken glass.
3. Needle-stick accidents can be reduced by (a) taking particular care, and (b) minimizing the use of
syringes and needles; for many techniques, syringes with blunt cannulas may be used instead.
Simple devices are available for opening septum-stoppered bottles so that pipettes can be used.
4. Needles should never be recapped. Without disconnecting them from the syringe (if available),
disposable articles should be discarded into puncture-proof containers fitted with covers.
5. Plastic Pasteur pipettes should replace those made of glass.
Separation of serum
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1. Only properly trained staff should be employed for this work.

2. Gloves and eye and mucous membrane protection should be worn.
3. Splashes and aerosols can only be avoided or minimized by good laboratory technique. Blood and
serum should be pipetted carefully, not poured. Pipetting by mouth must be forbidden.
4. After use, pipettes should be completely submerged in hypochlorite or other suitable disinfectant.
They should remain in the disinfectant for at least 18 h before disposal, or washing and sterilization
for reuse.
5. Discarded specimen tubes containing blood clots, etc. (with caps replaced) should be placed in
suitable leakproof containers for autoclaving and/or incineration.
6. A solution of hypochlorite, freshly prepared daily, should be available for clean-up of splashes and
spillages (see Chapter 14).
Use of centrifuges
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1. Satisfactory mechanical performance is a prerequisite of microbiological safety in the use of

laboratory centrifuges.
2. Centrifuges should be operated according to the manufacturers instructions.
3. Centrifuges should be placed at such a level that workers of less than average height can see into
the bowl to place trunnions and buckets correctly.
4. Centrifuge tubes and specimen containers for use in the centrifuge should be made of thick-walled
glass or preferably of plastic and should be inspected for defects before use.
5. Tubes and specimen containers should always be securely capped (screw-capped if possible) for
centrifugation.
6. The buckets must be loaded, equilibrated, sealed and opened in a biological safety cabinet.
7. Buckets and trunnions should be paired by weight and, with tubes in place, correctly balanced.
8. The amount of space that should be left between the level of the fluid and the rim of the centrifuge
tube should be given in manufacturers instructions.
9. Distilled water or alcohol (propanol, 70%) should be used for balancing empty buckets. Saline or
hypochlorite solutions should not be used as they corrode metals.
10. Sealable centrifuge buckets (safety cups) must be used for microorganisms of Risk Groups 3 and 4.
11. When using angle head centrifuge rotors, care must be taken to ensure that the tube is not
overloaded as it might leak.
12. The interior of the centrifuge bowl should be inspected daily for staining or soiling at the level of the
rotor. If staining or soiling are evident then the centrifugation protocols should be re-evaluated.
13. Centrifuge rotors and buckets should be inspected daily for signs of corrosion and for hair-line cracks.
14. Buckets, rotors and centrifuge bowls should be decontaminated after each use.
15. After use, buckets should be stored in an inverted position to drain the balancing fluid.
16. Infectious airborne particles may be ejected when centrifuges are used. These particles travel at
speeds too high to be retained by the cabinet air flow if the centrifuge is placed in a traditional open-
47
fronted Class I or Class II biological safety cabinet. Enclosing centrifuges in Class III safety cabinets
prevents emitted aerosols from dispersing widely. However, good centrifuge technique and securely
capped tubes offer adequate protection against infectious aerosols and dispersed particles.
Use of homogenizers, shakers, blenders and sonicators
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1. Domestic (kitchen) homogenizers should not be used in laboratories as they may leak or release
aerosols. Laboratory blenders and stomachers are safer.
2. Caps and cups or bottles should be in good condition and free from flaws or distortion. Caps should
be well-fitting and gaskets should be in good condition.
3. Pressure builds up in the vessel during the operation of homogenizers, shakers and sonicators.
Aerosols containing infectious materials may escape from between the cap and the vessel. Plastic, in
particular, polytetrafluoroethylene (PTFE) vessels are recommended because glass may break,
releasing infectious material and possibly wounding the operator.
4. When in use, homogenizers, shakers and sonicators should be covered by a strong transparent
plastic casing. This should be disinfected after use. Where possible, these machines should be
operated, under their plastic covers, in a biological safety cabinet.
5. At the end of the operation the containers should be opened in a biological safety cabinet.
6. Hearing protection should be provided for people using sonicators.
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Use of tissue grinders
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1. Glass grinders should be held in a wad of absorbent material in a gloved hand. Plastic (PTFE)
grinders are safer.
2. Tissue grinders should be operated and opened in a biological safety cabinet.
Care and use of refrigerators and freezers
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1. Refrigerators, deep-freezers and solid carbon dioxide (dry-ice) chests should be defrosted and
cleaned periodically, and any ampoules, tubes, etc. that have broken during storage removed. Face
protection and heavy duty rubber gloves should be worn during cleaning. After cleaning, the inner
surfaces of the cabinet should be disinfected.
2. All containers stored in refrigerators, etc. should be clearly labelled with the scientific name of the
contents, the date stored and the name of the individual who stored them. Unlabelled and obsolete
materials should be autoclaved and discarded.
3. An inventory must be maintained of the freezers contents.
4. Flammable solutions must not be stored in a refrigerator unless it is explosion-proof. Notices to this
effect should be placed on refrigerator doors.
Opening of ampoules containing lyophilized infectious materials

Care should be taken when ampoules of freeze-dried materials are opened, as the contents may be
under reduced pressure and the sudden inrush of air may disperse some of the materials into the
atmosphere. Ampoules should always be opened in a biological safety cabinet. The following procedures
are recommended for opening ampoules.
1.
2.
3.
4.
5.
6.
First decontaminate the outer surface of the ampoule.

Make a file mark on the tube near to the middle of the cotton or cellulose plug, if present
Hold the ampoule in a wad of alcohol-soaked cotton to protect hands before breaking it at a file
scratch.
Remove the top gently and treat as contaminated material.
If the plug is still above the contents of the ampoule, remove it with sterile forceps.
Add liquid for resuspension slowly to the ampoule to avoid frothing.
48
Storage of ampoules containing infectious materials

Ampoules containing infectious materials should never be immersed in liquid nitrogen because cracked
or imperfectly sealed ampoules may break or explode on removal. If very low temperatures are required,
ampoules should be stored only in the gaseous phase above the liquid nitrogen. Otherwise, infectious
materials should be stored in mechanical deep-freeze cabinets or on dry ice. Laboratory workers should
wear eye and hand protection when removing ampoules from cold storage.
The outer surfaces of ampoules stored in these ways should be disinfected when the ampoules are
removed from storage.
Special precautions with blood and other body fluids, tissues and excreta
The precautions outlined below are designed to protect laboratory workers against infection by
bloodborne pathogens.
Collection, labelling and transport of specimens
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1. Universal precautions (3) should always be followed; gloves should be worn for all procedures.
2. Blood should be collected from patients and animals by trained staff.
3. For phlebotomies, conventional needle and syringe systems should be replaced by single-use safety
vacuum devices that allow the collection of blood directly into stoppered transport and/or culture
tubes, automatically disabling the needle after use.
4. The tubes should be placed in adequate containers for transport to the laboratory (see Chapter 12 for
transport requirements) and within the laboratory facility (see Chapter 10). Request forms should be
placed in separate water-proof bags or envelopes.
5. Reception staff should not open these bags.
Containment
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1. Diagnostic work may be done in a basic laboratory - Biosafety Level 2, preferably one dedicated for
this purpose.
2. Research and development work involving propagation of large volumes or high concentrations of
infectious microorganisms may require a containment laboratory - Biosafety Level 3 or higher
containment level.
Opening specimen tubes and sampling contents
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1. Specimen tubes should be opened in a Class I or Class II biological safety cabinet.

2. Gloves must be worn. Eye and mucous membrane protection is also recommended (goggles or
shield (visor)).
3. Protective clothing should be supplemented with a plastic apron.
4. The stopper should be grasped through a piece of paper or gauze to prevent splashing.
Glass and sharps

1. Plastics should replace glass wherever possible. Only laboratory grade (borosilicate) glass should be
used, and any article that is chipped or cracked should be discarded.
2. Hypodermic needles must not be used as pipettes. Blunt cannulas are permitted.
Films and smears for microscopy

Fixing and staining of blood, sputum and faecal samples for microscopy does not necessarily kill all
organisms or viruses on the smears. These items should be handled with forceps, stored appropriately,
and decontaminated and/or autoclaved before disposal.
Automated equipment (sonicators, vortex mixers)

1. Equipment should be of the closed type to avoid dispersion of droplets and aerosols.
2. Effluents should be collected in closed vessels for further autoclaving and/or disposal.
49
3. Equipment should be disinfected at the end of each session, following the manufacturers
instructions.
Tissues
1. Formalin fixatives should be used. Small specimens, e.g. from needle biopsies, can be fixed and
decontaminated within a few hours, but larger specimens may take several days.
2. Frozen sectioning should be avoided. Should it be essential, the cryostat should be shielded and the
operator should wear a safety shield (visor). For decontamination, the temperature of the instrument
should be raised to 20 C.
Decontamination
Precautions with materials that may contain prions
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Hypochlorites and high-level disinfectants are recommended for decontamination. Freshly prepared
hypochlorite solutions should contain available chlorine at 1 g/l for general use and 10 g/l for blood
spillages. Glutaraldehyde may be used for decontaminating surfaces (see Chapter 14).
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Prions (also referred to as slow viruses) are associated with certain transmissible spongiform
encephalopathies (TSEs), notably Creutzfeldt-Jakob disease (CJD; including the new variant form),
Gerstmann-Strussler-Scheinker syndrome, fatal familial insomnia and kuru in humans; scrapie in sheep
and goats; bovine spongiform encephalopathy (BSE) in cattle; and other transmissible encephalopathies
of deer, elk and mink. Although CJD has been transmitted to humans, there appear to be no proven
cases of laboratory-associated infections with any of these agents. Nevertheless, it is prudent to observe
certain precautions in the handling of material from infected or potentially infected humans and animals.
The selection of a biosafety level for work with materials associated with TSEs will depend on the
samples to be studied, and should be undertaken in consultation with national authorities. The highest
concentrations of prions are found in central nervous system tissue. Animal studies suggest that it is
likely that high concentrations of prions are also found in the spleen, thymus, lymph nodes and lung.
As there is no method that will ensure decontamination after exposure to prions, it is important to
stress the use of disposable instruments whenever possible, and to use a protective covering for the work
surface of the biological safety cabinet, which can also be disposed of after use.
The main precaution to be taken is to avoid ingestion of contaminated materials or puncture of the
laboratory workers skin. The following additional precautions should be taken, as the agents are not
killed by the normal processes of laboratory disinfection and sterilization.
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1. The use of dedicated equipment, i.e. equipment not shared with other laboratories, is highly
recommended.
2. Disposable laboratory protective clothing (gowns and aprons) and gloves must be worn (steel mesh
gloves between rubber gloves for pathologists).
3. Use of disposable plastic ware, which can be treated and discarded as dry waste, is highly
recommended.
4. Tissue processors should not be used because of the problems of disinfection. Jars and beakers
should be used instead.
5. All manipulations must be conducted in biological safety cabinets.
6. Great care should be exercised to avoid aerosol production, accidental ingestion, and cuts and
punctures of the skin.
7. Formalin-fixed tissues should be regarded as still infectious, even after prolonged exposure to
formalin.
8. Bench waste, including disposable gloves, gowns and aprons, should be autoclaved, followed by
incineration.
9. Non-disposable instruments, including steel mesh gloves, must be collected for decontamination.
10. Infectious liquid waste contaminated with prions should be treated with 2 mol/l sodium hydroxide
(final concentration) for 1 h followed by autoclaving
11. Paraformaldehyde vaporization procedures do not diminish prion titres and prions are resistant to
ultraviolet irradiation. However, the cabinets must continue to be decontaminated by standard
methods (i.e. formaldehyde gas) to inactivate other agents that may be present.
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12. Prion-contaminated biological safety cabinets and other surfaces can be decontaminated by repeated
wetting with 2 mol/l sodium hydroxide for 1 h followed by rinsing with water. High-efficiency
particulate air (HEPA) filters should be autoclaved and incinerated at regular intervals.
13. Instruments should be soaked for 1 h in 2 mol/l sodium hydroxide and then rinsed well in water
before autoclaving.
14. Instruments that cannot be autoclaved can be cleaned by repeated wetting with 2 mol/l sodium
hydroxide over a 1-h period. Appropriate washing to remove residual sodium hydroxide is required.
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11. Biosafety and recombinant DNA technology
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Recombinant DNA technology involves combining genetic information from different sources thereby
creating genetically modified organisms (GMOs) that may have never existed in nature before. Initially
there was concern among molecular biologists that such organisms might have unpredictable and
undesirable properties and would represent a biohazard if they escaped from the laboratory. This
concern resulted in the famous Asilomar conference held in 1975 (24). At that meeting safety issues were
discussed and the first guidelines for recombinant DNA technology were proposed. More than 25 years
have now passed and no adverse incidents associated with this technology have been revealed. This
demonstrates that genetic engineering is safe, provided that appropriate safety measures are observed.
Recombinant DNA technology or genetic engineering was first used to clone DNA segments of
interest in bacterial hosts in order to produce enough sufficiently pure materials for further studies. More
recently, recombinant DNA molecules have also been used to create genetically modified higher
organisms such as transgenic and knock-out animals and transgenic plants (see the relevant sections
below).
Recombinant DNA technology has already had an enormous impact on biology and medicine and will
have an even greater influence in the near future. Now that the nucleotide sequence of the entire human
genome is available, tens of thousands of genes of unknown functions will be studied, and recombinant
DNA technology will be one of the means of doing so. Furthermore, gene therapy is expected to become
an accepted treatment for certain diseases in the future, and many new vectors for gene transfer will be
devised using genetic engineering techniques. Finally, transgenic plants produced by recombinant DNA
technology may play an increasingly important role in modern agriculture.
When considering the use or construction of GMOs, the risk assessment process for work in the
laboratory is perhaps even more important than that for work with genetically normal (non-modified)
organisms. Whereas the latter are likely to be well-characterized with respect to pathogenic properties,
the former will be novel, and evaluation of the potential hazards associated with working with such
organisms cannot build on experience only.
The risk assessment will identify the biological containment system to be used. The properties of the
donor organism, the nature of the DNA sequences that will be transferred, the properties of the recipient
organism, and the properties of the environment must be evaluated. All of these factors will determine the
biosafety level that is required for the safe handling of the resulting GMO. The following paragraphs
provide some background information with respect to these criteria.
Biological expression systems

Biological expression systems are vectors and host cells that fulfil a number of criteria that make them
safe to use. A good example of a biological expression system is plasmid pUC18 (or derivatives thereof),
which is frequently used as a cloning vector in combination with Escherichia coli K12 cells. The pUC18
plasmid and its derivatives have been entirely sequenced. More importantly, all genes required for
efficient transfer to other bacteria have been deleted from the precursor plasmid pBR322 providing
significant containment. E. coli K12 is a strain that lacks the genes known to render some E. coli strains
pathogenic. Furthermore, E. coli K12 cannot permanently colonize the gut of healthy humans or animals.
Thus, most routine genetic engineering experiments can be performed safely in E. coli K12/pUC18 at
Biosafety Level 1 provided the inserted foreign DNA sequences do not require a higher biosafety level
(see below).
Properties of the donor organism and cloned DNA

Risk assessment must consider not only the vector/host system used but also the properties of the DNA
to be cloned. In most cases the risk assessment will show that the inserted DNA sequences are unlikely
to alter the biological properties of the host organism, but in some cases they may do so, for example, if
they are derived from a pathogenic organism. Obviously not all genes of a pathogenic organism
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Biosafety and recombinant DNA technology
contribute to the virulence of the agent. Therefore, insertion of well-characterized DNA sequences that
are unlikely to be involved in pathogenicity may not require additional safety measures. However, in
cases where these sequences are not characterized, a situation that is typically encountered when a
library of genomic DNA of an organism is being established, a higher biosafety level will be required.
An important consideration is whether the gene product has potential pharmacological activity.
Cloning of genes coding for proteins such as toxins may therefore require higher biosafety levels. Overexpression of gene products from eukaryotic viral vectors can have unexpected consequences when
these proteins have pharmacological activity.
Viral vectors for gene transfer
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Viral vectors are used not only for gene therapy but also for efficient transfer of genes to other cells.
Adenovirus vectors have become popular for gene therapy. Such vectors lack certain genes that are
required for virus replication and therefore have to be propagated in cell lines that complement the defect.
Although such vectors are replication-defective, they should be handled at the same biosafety level as
the parent adenovirus from which they are derived. The reason for this is that the virus stocks may be
contaminated with replication-competent viruses, which are generated by rare spontaneous
recombination events in the complementing cell line.
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Transgenic and knock-out animals
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Animals carrying foreign genetic information (transgenic animals) should be handled in containment
levels appropriate to the characteristics of the products of the foreign genes. Animals with targeted
deletions of specific genes (knock-out animals) do not generally present particular biological hazards.
Examples of transgenic animals include animals expressing receptors for viruses normally unable to
infect that species. If such animals escaped from the laboratory and transmitted the transgene to the wild
animal population, an animal reservoir for that particular virus could theoretically be generated.
This possibility has been discussed for poliovirus and is particularly relevant in the context of
poliomyelitis eradication.
Transgenic mice expressing the human poliovirus receptor generated in different laboratories were
susceptible to poliovirus infection by various inoculation routes and the resulting disease was clinically
and histopathologically similar to human poliomyelitis. However, the mouse model differs from humans in
that alimentary tract replication of orally administered poliovirus is either inefficient or does not occur. It is
therefore very unlikely that escape of such transgenic mice to the wild would result in the establishment
of a new animal reservoir for poliovirus. Nevertheless, this example indicates that, for each new line of
transgenic animal, detailed studies should be conducted to determine the routes by which the animals
can be infected, the inoculum size required for infection, and the extent of virus shedding by the infected
animals. In addition, all measures should be taken to assure strict containment of receptor transgenic
mice.
Transgenic plants
Transgenic plants expressing genes that confer tolerance to herbicides or resistance to insects are
currently a matter of considerable controversy in large parts of the world. The discussions mainly focus
on the safety of such plants as food and on the long-term ecological consequences of growing such
plants on a large scale, which are not the subjects of this chapter.
Transgenic plants expressing genes of animal or human origin should remain strictly contained within
the facility. Such transgenic plants should be handled at biosafety levels appropriate to the characteristics
of the products of the expressed genes.
Conclusions
When creating or handling recombinant organisms, it is essential to perform a detailed risk assessment,
which must take into account the nature of the donor, the recipient organism and the environment. In
many cases the risk assessment will show that the recombinant organism can be handled at the same
biosafety level as the wild-type recipient. In some instances, however, higher biosafety levels will be
required. This is the case, for example, when ill-defined DNA sequences from a donor organism are
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transferred, which could potentially increase the virulence of the recipient organism. This situation is
typically encountered in random (shot-gun) cloning experiments in which genomic DNA libraries are
established. Risk assessment is particularly important when creating GMOs expressing proteins with
pharmacological activity, such as toxins. It is obvious that such organisms must be handled with caution.
Some pharmacologically active proteins are only toxic when expressed at high levels. In this case, the
risk assessment becomes very demanding and requires an estimation of the expected expression levels
of the protein by a particular recombinant organism and the levels at which a given protein becomes toxic
in an organism accidentally exposed to it. Many countries have national safety committees, which
establish guidelines for work with GMOs and help scientists classify their work at the appropriate
biosafety level. In some cases classification may differ between countries, or countries may decide to
classify work at a lower or higher level when new information on a particular vector/host system becomes
available. Risk assessment is thus a dynamic process and has to take into account new developments
and the progress of science. It is the responsibility of the scientists involved in genetic engineering to
keep up to date on these developments, and to respect the guidelines established by their national safety
committee.
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For further information see references 25-27.
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12. Transport of infectious substances
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International regulations for the transport of infectious materials are based on the biennial
recommendations of the United Nations Committee of Experts on the Transport of Dangerous Goods. In
December 2002, at the end of the current biennium 2001-2002, the Committee may introduce further
changes to a proposal to amend the current requirements in the United Nations Transport of Dangerous
Goods Model Regulations that has been recently accepted at a biannual session. The proposal that
has been adopted, and the further changes that may be introduced, will substantially affect the current
regulations concerning the transport of infectious substances. Chapter 12 on Transport of infectious
substances is therefore awaiting the final vote to be taken in December 2002 by the Committee.
The Laboratory biosafety manual, 3rd ed., which will include the revised Chapter 12, will be made
available on the WHO web site and published in 2003.
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13. Contingency plans and

emergency procedures
Every laboratory that works with infective microorganisms should institute safety precautions appropriate
to the hazard of the organisms and the animals being handled.
A written contingency plan for dealing with laboratory and animal facility accidents is a necessity in
any facility that works with or stores Risk Group 3 or 4 microorganisms (containment laboratory
Biosafety Level 3 and maximum containment laboratory Biosafety Level 4). National and/or local health
authorities should be involved in the development of the emergency preparedness plan.
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Contingency plan
The contingency plan should provide operational procedures for:
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precautions against natural disasters, e.g. fire, flood, earthquake, and explosion
biohazard risk assessment
accident-exposure management and decontamination
emergency evacuation of people and animals from the premises
emergency medical treatment of exposed and injured persons
medical surveillance of exposed persons
clinical management of exposed persons
epidemiological investigation.
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In the development of this plan the following items should be considered for inclusion:
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identification of high-risk organisms

location of high-risk areas, e.g. laboratories, storage areas, animal facilities
identification of at-risk personnel and populations
identification of responsible personnel and their duties, e.g. biosafety officer, safety personnel, local
health authority, clinicians, microbiologists, veterinarians, epidemiologists, and fire and police
services
lists of treatment and isolation facilities that can receive exposed or infected persons
transport of exposed or infected persons
lists of sources of immune serum, vaccines, drugs, special equipment and supplies
provision of emergency equipment, e.g. protective clothing, disinfectants, decontamination
equipment.
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Emergency procedures for microbiological laboratories

Accidental injection, cuts and abrasions
The affected individual should remove protective clothing, wash the hands and the affected part, apply an
appropriate skin disinfectant, go to the first-aid room, and inform the person in charge about the cause of
the wound and the organisms involved. If considered necessary, a physician should be consulted and his
or her advice followed. Appropriate and complete medical records should be kept.
Accidental ingestion of potentially hazardous material

Protective clothing should be removed and the individual taken to the first-aid room. A physician should
be informed of the material ingested and his or her advice followed. Appropriate and complete medical
records should be kept.
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Contingency plans and emergency procedures
Potentially hazardous aerosol release (other than in a biological safety cabinet)

All persons should immediately vacate the affected area and any exposed persons should be referred for
medical advice. The laboratory supervisor and the biosafety officer should be informed at once. No one
should enter the room for at least 1 h, to allow aerosols to be carried away and heavier particles to settle.
If the laboratory does not have a central air exhaust system, entrance should be delayed for 24 hSigns
should be posted indicating that entry is forbidden. After the appropriate time, decontamination should
proceed, supervised by the biosafety officer. Appropriate protective clothing and respiratory protection
should be worn for this.
Broken and spilled infectious substances, including cultures
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Broken objects contaminated with infectious substances, including vials or containers, or spilled
infectious substances, including cultures, should be covered with a cloth or paper towels. Disinfectant
should then be poured over these and left for at least 30 min. The cloth or paper towels and the broken
material may then be cleared away; glass fragments should be handled with forceps. The contaminated
area should then be swabbed with disinfectant. If dustpans are used to clear away the broken material,
they should be autoclaved or placed in an effective disinfectant for 24 h. Cloths, paper towels and swabs
used for cleaning up should be placed in a contaminated-waste container. Gloves should be worn for all
these procedures.
If laboratory forms or other printed or written matter are contaminated, the information should be
copied onto another form and the original discarded into the contaminated-waste container.
Breakage of tubes containing potentially hazardous material in centrifuges not having
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If a breakage occurs or is suspected while the machine is running, the motor should be switched off and
the machine left closed for 30 min. If a breakage is discovered after the machine has stopped, the lid
should be replaced immediately and left closed for 30 min. In both instances, the biosafety officer should
be informed.
Strong (e.g. thick rubber) gloves, covered if necessary with suitable disposable gloves, should be
worn for all subsequent operations. Forceps, or cotton held in the forceps, should be used to retrieve
glass debris.
All broken tubes, glass fragments, buckets, trunnions and the rotor should be placed in noncorrosive
disinfectant known to be active against the organisms concerned and left for 24 h and/or autoclaved.
Unbroken, capped tubes may be placed in disinfectant in a separate container and recovered after
60 min.
The centrifuge bowl should be swabbed with the same disinfectant, at the appropriate dilution, left
overnight and then swabbed again, washed with water and dried. All materials used in the clean-up
should be treated as infectious waste.
Breakage of tubes inside sealable buckets (safety cups)

All sealed centrifuge buckets should be loaded and unloaded in a biological safety cabinet. If a breakage
is suspected, the cap should be opened and left loose and the bucket autoclaved.
Fire, flood and natural disaster

Fire and other services should be involved in the development of emergency plans. They should be told
in advance which rooms contain potentially infectious materials. It is beneficial to arrange for these
services to visit the laboratory to become acquainted with its layout and contents if possible.
After a flood or other natural disaster (including earthquake), local or national emergency services
should be warned of the potential hazards within and/or near laboratory buildings. They should enter only
when accompanied by a trained laboratory worker. Cultures and infectious materials should be collected
in leakproof boxes or strong disposable bags. Salvage or final disposal should be determined by safety
staff on the basis of local knowledge.
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Vandalism
Vandalism is usually selective (e.g. aimed at animal houses). Suitable defences are strong, heavy doors,
good locks and restricted entry. Screened windows and intruder alarms are desirable. Action after
vandalism is the same as that for other emergencies.
Emergency services: whom to contact

The telephone numbers and addresses of the following should be prominently displayed near all
telephones:
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the institution or laboratory itself (the address and location may not be known in detail by the caller or
the services called)
director of the institution or laboratory
laboratory supervisor
biosafety officer
fire services
hospital/ambulance service (if a particular hospital has arranged to accept casualties, e.g. high-risk
personnel, the names of individual departments and doctors)
police
medical officer
responsible technician
water, gas and electricity services.
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Emergency equipment
first-aid kit, including universal and special antidotes

stretcher
appropriate fire extinguishers, fire blankets
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full protective clothing (one-piece coveralls, gloves and head covering for incidents involving
microorganisms in Risk Groups 3 and 4)
full-face respirators with appropriate chemical and particulate filter canisters
room disinfection apparatus, e.g. sprays and formaldehyde vaporizers
tools, e.g. hammers, axes, spanners, screwdrivers, ladders, ropes
hazard area demarcation equipment and notices.
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The following are also suggested but may be varied according to local circumstances:
For further information see references 12 and 28.
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14. Disinfection and sterilization
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A basic knowledge of disinfection and sterilization is crucial for biosafety in the laboratory. Since heavily
soiled items cannot promptly be efficiently disinfected or sterilized, it is equally important to understand
the fundamentals of precleaning. In this regard, the following general principles apply to all known
classes of microbial pathogens, with the notable exception of prions, which are dealt with separately in
this chapter.
The specific requirements for decontamination for biosafety will depend on the type of experimental
work and the nature of the infectious agent(s) being handled. It will, therefore, be necessary to use the
generic information given here to develop more specific and standardized procedures to suit the needs of
the various levels of biohazard involved in a particular laboratory.
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Definitions
Disinfectant
Disinfection
Microbicide
Sterilization
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Biocide
Chemical germicide
Decontamination
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Antiseptic
An agent that kills microorganisms or suppresses their growth and

multiplication.
A substance that inhibits the growth and development of microorganisms
without necessarily killing them. Antiseptics are usually applied on body
surfaces.
A general term for any agent that kills unicellular and multicellular organisms.
A chemical or a mixture of chemicals used to kill microorganisms.
Any process for removing and/or killing microorganisms. The same term is
also used for removing or neutralizing hazardous chemicals and radioactive
materials.
A chemical or mixture of chemicals used to kill microorganisms, but not
necessarily their spores. Disinfectants are usually applied on inanimate
surfaces or objects.
A physical or chemical means of killing microorganisms, but not necessarily
their spores.
A chemical or mixture of chemicals that kills microorganisms. The term is
often used in place of biocide, germicide or antimicrobial.
A process that destroys and/or removes all classes of microorganisms and
their spores.
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Antimicrobial
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Many different terms are used in dealing with the process of decontamination for biosafety. The following
are among the more common.
Precleaning and cleaning laboratory materials

In practical terms, cleaning is the removal of visible dirt and stains. This is generally achieved either by
(a) brushing, vacuuming or dry dusting; or (b) washing or damp mopping with water containing a soap or
detergent. Where the risk of human or animal contact with pathogen-contaminated materials is high and
subsequent decontamination is needed, precleaning is routinely carried out. This is necessary because
dirt and soil can shield microorganisms and can also interfere with the killing action of chemical
germicides. In such cases, precleaning is essential to achieve proper disinfection or sterilization. Also,
many germicidal products claim activity only on precleaned items. Precleaning must be carried out with
care to avoid exposure to infectious agents, and materials chemically compatible with the germicides to
be applied later must be used. It is quite common to use the same chemical germicide for precleaning
and disinfection.
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Chemical germicides
Table 10. Recommended dilutions of chlorine-releasing compounds
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Many types of chemicals can be used as disinfectants and antiseptics and there is an ever-increasing
number and variety of commercial products. Formulations must therefore be carefully selected for
specific needs, and stored, used and disposed of as directed by the manufacturer. The germicidal activity
of many chemicals is faster and better at higher temperatures. At the same time, higher temperatures can
accelerate their evaporation and also degrade them faster. Particular care is needed in the use and
storage of such chemicals in tropical regions, where their shelf-life may be reduced because of high
ambient temperatures.
Many germicides can be harmful to humans and the environment. They should therefore be selected,
handled and disposed of with care. For personal safety, gloves, aprons and eye protection are
recommended when preparing use-dilutions of chemical germicides. Chemical germicides are therefore
not required for regular and general cleaning of floors, walls, equipment and furniture except in cases of
outbreak control.
Commonly used classes of chemical germicides are described below, with generic information on their
applications and safety profiles. Unless otherwise indicated, the germicide concentrations are given in
weight/volume (w/v). Table 10 summarizes the recommended dilutions of chlorine-releasing compounds.
Available chlorine required
0.1% (1 g/l)
0.5% (5 g/l)
Sodium hypochlorite solution (5% available chlorine)
20 ml/l
100 ml/l
Calcium hypochlorite (70% available chlorine)
1.4 g/l
7.0 g/l
1.7 g/l
8.5 g/l
1 tablet per litre
4 tablets per litre
20 g/l
20 g/l
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For flooding, e.g. on blood or before removal of bulk material.
See text.
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Sodium dichloroisocyanurate tablets

(1.5 g available chlorine per tablet)
Chloramine (25% available chlorine)
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Dirty conditions
Sodium dichloroisocyanurate powder

(60% available chlorine)
Clean conditions
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Chlorine (sodium hypochlorite)

Chlorine, a fast-acting oxidant, is a widely available and broad-spectrum germicide. It is normally sold as
bleach, an aqueous solution of sodium hypochlorite (NaOCl), which can be diluted with water to provide
various concentrations of available chlorine.
Chlorine, especially as bleach, is highly alkaline and can be corrosive to metal. Its activity is
considerably reduced by organic matter (protein). Storage of stock or working solutions of bleach in open
containers, particularly at high temperatures, releases chlorine gas thus weakening their germicidal
potential. The frequency with which working solutions of bleach should be changed depends on their
starting strength, the type (e.g. with or without a lid) and size of their containers, the frequency and nature
of use, and ambient conditions. As a general guide, solutions receiving materials with high levels of
organic matter several times a day should be changed at least daily, while those with less frequent use
may last for as long as a week.
A general all-purpose laboratory disinfectant should have a concentration of 1 g/l available chlorine. A
stronger solution, containing 5 g/l available chlorine is recommended for dealing with biohazardous
spillage and in the presence of large amounts of organic matter. Sodium hypochlorite containing 5 g/l
available chlorine is recommended as the disinfectant of choice in emergency situations involving viruses
such as Hantavirus, and Lassa and Ebola viruses.
Sodium hypochlorite solutions, as domestic bleach, contain 50 g/l available chlorine and should
therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l, respectively. Industrial
solutions of bleach have a sodium hypochlorite concentration of nearly 120 g/l and must be diluted
accordingly to obtain the levels indicated above.
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Disinfection and sterilization
Granules or tablets of calcium hypochlorite (Ca(ClO)2) generally contain about 70% available chlorine.
Solutions prepared with granules or tablets, containing 1.4 g/l and 7.0 g/l, will then contain 1.0 g/l and
5 g/l available chlorine, respectively.
Bleach is not recommended as an antiseptic, but may be used as a general-purpose disinfectant and
for soaking contaminated metal-free materials. In emergencies, bleach can also be used to disinfect
water for drinking with a final concentration of 12 mg/l available chlorine.
Note. Chlorine gas is highly toxic. Bleach must therefore be stored and used in well-ventilated areas only.
Also, bleach must not be mixed with acids in order to avoid the rapid release of chlorine gas. Many byproducts of chlorine can be harmful to humans and the environment, so that indiscriminate use of
chlorine-based disinfectants, and in particular bleach, should be avoided.
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Sodium dichloroisocyanurate
Sodium dichloroisocyanurate (NaDCC) in powder form contains 60% available chlorine. Solutions
prepared with NaDCC powder at 1.7 g/l and 8.5 g/l will contain 1 g/l or 5 g/l available chlorine,
respectively. Tablets of NaDCC generally contain the equivalent of 1.5 g available chlorine per tablet.
One or four tablets dissolved in 1 litre of water will give the required concentrations of 1 g/l or 5 g/l,
respectively. NaDCC as powder or tablets is easy and safe to store. Solid NaDCC can be applied on
spills of blood or other biohazardous liquids and left for at least 10 min before removal. Further cleaning
of the affected area can then take place.
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Chloramines
Chloramines are available as powders containing about 25% available chlorine. Chloramines release
chlorine at a slower rate than hypochlorites. Higher initial concentrations are therefore required for
efficiencies equivalent to those of hypochlorites. On the other hand, chloramine solutions are not
inactivated by organic matter to the same extent as hypochlorite solutions, and concentrations of 20 g/l
are recommended for both clean and dirty situations.
Chloramine solutions are virtually odour-free. However, items soaked in them must be thoroughly
rinsed to remove any residue of the bulking agents added to chloramine-T (sodium tosylchloramide)
powders. Chloramines can also be used to disinfect water for drinking when used at a final concentration
of 12 mg/l available chlorine.
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Chlorine dioxide
Chlorine dioxide is a strong and fast-acting germicide, often reported to be active at levels lower than
those needed by chlorine as bleach. To obtain an active solution for laboratory use it is generally
necessary to mix two separate components, hydrochloric acid (HCl) and sodium chlorite (NaClO2).
Stability can be an important issue with this germicide, and materials compatibility and corrosiveness
must also be considered when selecting products based on it.
Formaldehyde
Formaldehyde (HCHO) is a gas that kills all microorganisms and their spores, at temperatures above
20 C. Formaldehyde is not active against prions. It is relatively slow-acting and needs a relative humidity
level of about 70%. It is marketed as the solid polymer, paraformaldehyde, in flakes or tablets, or as
formalin, a solution of the gas in water of about 370 g/l (37%), containing methanol (100 ml/l) as a
stabilizer. Both formulations are heated to liberate the gas, which is used for decontamination and
disinfection of enclosed volumes such as safety cabinets and rooms (see section on Local environmental
decontamination, below).
Formaldehyde (5% formalin in water) may be used as a liquid disinfectant.
Note. Formaldehyde is a suspected carcinogen. It has a pungent smell and its fumes can irritate eyes
and mucous membranes. It must therefore be stored and used in a fume-hood or well-ventilated areas.
Applicable regulations on chemical safety must be consulted prior to its use.
Glutaraldehyde
Like formaldehyde, glutaraldehyde (OHC(CH2)3CHO) is also active against vegetative bacteria, spores,
fungi and lipid- and nonlipid-containing viruses. It is non-corrosive and faster acting than formaldehyde.
However, it takes several hours to kill bacterial spores. It is generally supplied as a solution with a
concentration of about 20 g/l (2%) and most products need to be activated (made alkaline) before use
by the addition of a bicarbonate compound supplied with the product. The activated solution can be
reused for 14 weeks depending on the formulation and type and frequency of its use. Dipsticks supplied
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with some products give only a rough indication of the levels of active glutaraldehyde available in
solutions under use. Glutaraldehyde solutions should be discarded if they become turbid.
Note. Glutaraldehyde is toxic and an irritant to skin and mucous membranes, and contact with it must be
avoided. It must be used in a fume-hood or in well-ventilated areas. It is not recommended as a spray or
solution for the decontamination of environmental surfaces. Applicable regulations on chemical safety
must be consulted prior to its use.
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Phenolic compounds
Phenolic compounds, a broad group of agents, were among the earliest germicides. However, results of
more recent safety concerns restrict their use. They are active against vegetative bacteria and lipidcontaining viruses and, when properly formulated, also show activity against mycobacteria. They are not
active against spores and their activity against nonlipid viruses is variable. Many phenolic products are
used for the decontamination of environmental surfaces, and some (e.g. triclosan and chloroxylenol) are
among the more commonly used antiseptics.
Triclosan is common in products for hand-washing. It is active mainly against vegetative bacteria and
safe for skin and mucous membranes. However, in laboratory-based studies, bacteria made resistant to
low concentrations of triclosan also show resistance to certain types of antibiotics. The significance of this
finding in the field remains unknown.
Note. Phenolic compounds are not recommended for use on food contact surfaces and in areas with
young children. They may be absorbed by rubber and can also penetrate the skin.
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Quaternary ammonium compounds

Many types of quaternary ammonium compounds are used as mixtures and often in combination with
other germicides, such as alcohols. They have good activity against vegetative bacteria and lipidcontaining viruses. Certain types (e.g. benzalkonium chloride) are used as antiseptics.
Note. The germicidal activity of certain types of quaternary ammonium compounds is considerably
reduced by organic matter, water hardness and anionic detergents. Care is therefore needed in selecting
agents for precleaning when quaternary ammonium compounds are to be used for disinfection.
Potentially harmful bacteria can grow in quaternary ammonium compound solutions. Owing to low
biodegradability, these compounds may also accumulate in the environment.
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Alcohols
Ethanol (ethyl alcohol, C2H5OH) and 2-propanol (isopropyl alcohol, (CH3)2CHOH) have similar
disinfectant properties. They are active against vegetative bacteria, fungi and lipid-containing viruses but
not against spores. Their action on nonlipid viruses is variable. For highest effectiveness they should be
used at concentrations of approximately 70% (v/v) in water: higher or lower concentrations may not be as
germicidal. A major advantage of aqueous solutions of alcohols is that they do not leave any residue on
treated items.
Mixtures with other agents are more effective than alcohol alone, e.g. 70% (v/v) alcohol with 100 g/l
formaldehyde, and alcohol containing 2 g/l available chlorine. A 70% (v/v) aqueous solution of ethanol
can be used on skin, work surfaces of laboratory benches and biosafety cabinets, and to soak small
pieces of surgical instruments. The contact time on skin should be no less than 10 s and that on
environmental surfaces no less than 3 min. Since ethanol can dry the skin, it is often mixed with
emollients. Alcohol-based hand-rubs are recommended for the decontamination of lightly soiled hands in
situations where proper hand-washing is inconvenient or not possible. However, it must be remembered
that ethanol is ineffective against spores and may not kill all types of nonlipid viruses.
Note. Alcohols are volatile and flammable and must not be used near open flames. Working solutions
should be stored in proper containers to avoid the evaporation of alcohols. Alcohols may harden rubber
and dissolve certain types of glue. Proper inventory and storage of ethanol in the laboratory is very
important to avoid its use for purposes other than disinfection. The availability and use of ethanol in
certain regions may be restricted owing to religious reasons. Bottles with alcohol-containing solutions
must be clearly labelled to avoid their accidental autoclaving.
Iodine and iodophors
The action of these disinfectants is similar to that of chlorine, although they may be slightly less inhibited
by organic matter. Iodine can stain fabrics and environmental surfaces and is generally unsuitable for use
as a disinfectant. On the other hand, iodophors and tinctures of iodine are good antiseptics. Polyvidoneiodine is a reliable and safe surgical scrub and preoperative skin antiseptic. Antiseptics based on iodine
are generally unsuitable for use on medical/dental devices. Iodine should not be used on aluminium or
copper.
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Note. Iodine can be toxic. Organic iodine-based products must be stored at 410 C to avoid the growth
of potentially harmful bacteria in them.
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Hydrogen peroxide and peracids

Like chlorine, hydrogen peroxide (H2O2) and peracids are strong oxidants and can be potent broadspectrum germicides. They are also safer than chlorine to humans and the environment.
Hydrogen peroxide is supplied either as a ready-to-use 3% solution or as a 30% aqueous solution to
be diluted to 510 times its volume with sterilized water. However, such 36% solutions of hydrogen
peroxide alone are relatively slow and limited as germicides. Products now available have other
ingredients to stabilize the hydrogen peroxide content, to accelerate its germicidal action and to make it
less corrosive.
Hydrogen peroxide can be used for the decontamination of work surfaces of laboratory benches and
biosafety cabinets, and stronger solutions may be suitable for disinfecting heat-sensitive medical/dental
devices. The use of vaporized hydrogen peroxide or peracetic acid (CH3COOOH) for the
decontamination of heat-sensitive medical/surgical devices requires specialized equipment.
Note. Hydrogen peroxide and peracids can be corrosive to metals such as aluminium, copper, brass and
zinc, and can also decolourize fabrics, hair, skin and mucous membranes. Articles treated with them
must be thoroughly rinsed before contact with eyes and mucous membranes. They should always be
stored away from heat and protected from light.
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Local environmental decontamination
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Decontamination of the laboratory space, its furniture and its equipment requires a combination of liquid
and gaseous disinfectants. Surfaces can be decontaminated using a solution of sodium hypochlorite
(NaOCl); a solution containing 1 g/l available chlorine may be suitable for general environmental
sanitation, but stronger solutions (5 g/l) are recommended when dealing with high-risk situations. For
environmental decontamination, formulated solutions containing 3% hydrogen peroxide (H2O2) make
suitable substitutes for bleach solutions.
Rooms and equipment can be decontaminated by fumigation with formaldehyde gas generated by
heating paraformaldehyde or boiling formalin. All openings in the room (i.e. windows, doors, etc.) should
be sealed with masking tape or similar before the gas is generated. Fumigation should be conducted at
an ambient temperature of at least 21 C and a relative humidity of 70%. (See also section on
Decontamination of biological safety cabinets, below.)
The gas should be in contact with the surfaces to be decontaminated for at least 8 h. After fumigation
the area must be ventilated thoroughly before personnel are allowed to enter. Appropriate respirators
must be worn by anyone entering the room before it has been ventilated. Gaseous ammonium
bicarbonate can be used to neutralize the formaldehyde.
Fumigation of spaces with vapours of solution of hydrogen peroxide has been reported but requires
further study.
Note. Formaldehyde is a dangerous and irritant gas and is a suspected carcinogen. Full-face respirators
with air supply may be necessary. The two-person rule should apply.
Decontamination of biological safety cabinets

To decontaminate Class I and Class II cabinets, the appropriate amount of paraformaldehyde (final
concentration of 0.8% paraformaldehyde in air) should be placed on an electric hot plate or fry pan
(controlled from outside the cabinet). A second hot plate or fry pan containing 10% more ammonium
bicarbonate than paraformaldehyde (with controls outside the cabinet) is also placed inside the cabinet.
This second pan should have a cover over it that can be removed remotely (e.g., attached to a string that
can be pulled from outside the cabinet). This will minimize premature neutralization of the formaldehyde
gas.
If the relative humidity is below 70%, an open container of hot water should also be placed inside the
cabinet before the front closure is sealed in place with strong tape (e.g., duct tape). If there is no front
closure, heavy gauge plastic sheeting is taped over the front to make sure that the gas cannot seep into
the room.
The switch for the paraformaldehyde pan is turned on, then off 1 h later, or when the
paraformaldehyde has all vaporized. The cabinet is left undisturbed overnight. The second pan is turned
on after the cover is removed and the ammonium bicarbonate is allowed to vaporize. At that time, the pan
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is turned off and the cabinet is turned on and allowed to circulate the ammonium bicarbonate gas for 1 h.
The front closure (or plastic sheeting) can then be removed and the cabinet used.
Hand-washing/hand decontamination
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Whenever possible, suitable gloves should be worn when handling biohazardous materials. However,
this does not replace the need for regular and proper hand-washing by laboratory personnel. Hands must
be washed after handling biohazardous materials and animals, and using the toilet, and before leaving
the laboratory, and eating.
In most situations, thorough washing of hands with ordinary soap and water is sufficient to
decontaminate them, but the use of germicidal soaps is recommended in high-risk situations. Hands
should be thoroughly lathered with soap, using friction, for at least 10 s, rinsed in clean water and dried
using a clean paper or cloth towel (if available, warm-air hand-dryers are also recommended).
Foot- or elbow-operated faucets are recommended. Where not fitted, a paper/cloth towel should be
used to turn off the faucet handles to avoid recontaminating washed hands.
As mentioned above, alcohol-based hand-rubs should be used to decontaminate lightly soiled hands
when proper hand-washing is not available or not convenient.
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Heat disinfection and sterilization
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Heat is the most common among the physical agents used for the decontamination of pathogens. Dry
heat, which is totally non-corrosive, is used to process many items of laboratory ware which can
withstand temperatures of 160 C or higher for 24 h. Burning or incineration (see below) is also a form of
dry heat. Moist heat is most effective when used in the form of autoclaving.
Boiling does not necessarily kill all microorganisms and/or pathogens, but it may be used as the
minimum processing for disinfection where other methods (chemical disinfection or decontamination,
autoclaving) are not applicable or available.
Sterilized items must be handled and stored such that they remain uncontaminated until used.
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Autoclaving
3 min holding time at 134 C

25 min holding time at 115 C.
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Saturated steam under pressure (autoclaving) is the most effective and reliable means of sterilizing
laboratory materials. For most purposes, the following cycles will ensure sterilization of correctly loaded
autoclaves:
Examples of different autoclaves include the following.

Gravity displacement autoclaves. Fig. 7 shows the general construction of a gravity-displacement
autoclave. Steam enters the chamber under pressure and displaces the heavier air downwards and
through the valve in the chamber drain, fitted with a HEPA filter.
Pre-vacuum autoclaves. These machines allow the removal of air from the chamber before steam is
admitted. The exhaust air is evacuated through a valve fitted with a HEPA filter. At the end of the cycle,
the steam is automatically exhausted. These autoclaves can operate at 134 C and the sterilization cycle
can therefore be reduced to 3 min. They are ideal for porous loads, but cannot be used to process liquids
because of the vacuum.
Fuel-heated pressure cooker autoclaves. These should be used only if a gravity displacement autoclave
is not available. They are loaded from the top and heated by gas, electricity or other types of fuels. Steam
is generated by heating water in the base of the vessel and air is displaced upwards through a relief vent.
When all the air has been removed, the valve on the relief vent is closed and the heat reduced. The
pressure and temperature rise until the safety valve operates at a preset level. This is the start of the
holding time. At the end of the cycle the heat is turned off and the temperature allowed to fall to 80 C or
below before the lid is opened.
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Loading autoclaves
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Fig. 7. Gravity displacement autoclave
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Materials should be loosely packed in the chamber for easy steam penetration and air removal. Bags
should allow the steam to reach their contents.
Precautions in the use of autoclaves
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The following rules can minimize the hazards inherent in operating pressurized vessels.
1. Responsibility for operation and routine care should be assigned to trained individuals and a
preventive maintenance programme should include regular inspection of the chamber, door seals
and all gauges and controls by qualified personnel.
2. The steam should be saturated and free from corrosion inhibitors or other chemicals, which could
contaminate the items being sterilized.
3. All materials to be autoclaved should be in containers that allow ready removal of air and permit good
heat penetration; the chamber should not be tightly packed or steam will not reach the load evenly.
4. For autoclaves without an interlocking safety device that prevents the door being opened when the
chamber is pressurized, the main steam valve should be closed and the temperature allowed to fall
below 80 C before the door is opened.
5. Operators should wear suitable gloves and visors for protection when opening the autoclave, even
when the temperature has fallen below 80 C.
6. In any routine monitoring of autoclave performance, biological indicators or thermocouples should be
placed at the centre of each load. Regular monitoring with thermocouples and recording devices in a
worst case load is highly desirable to determine proper operating cycles.
7. The drain screen filter of the chamber (if available) should be removed and cleaned daily.
8. Care should be taken to ensure that the relief valves of pressure cooker autoclaves do not become
blocked by paper, etc. in the load.
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Incineration
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Incineration is useful for disposing of animal carcasses as well as anatomical and other laboratory waste
with or without prior decontamination (see section on Contaminated (infectious) materials for disposal in
Chapter 3). Incineration of infectious materials is an alternative to autoclaving only if the incinerator is
under laboratory control, and it is provided with an efficient means of temperature control and a
secondary burning chamber.
Many incinerators, especially those with a single combustion chamber, are unsatisfactory for dealing
with infectious materials, animal carcasses and plastics. Such materials may not be completely destroyed
and the effluent from the chimney may pollute the atmosphere with microorganisms, toxic chemicals and
smoke. However, there are many satisfactory configurations for combustion chambers. Ideally the
temperature in the primary chamber should be at least 800 C and that in the secondary chamber at least
1000 C. Materials for incineration, even with prior decontamination, should be transported to the
incinerator in bags, preferably plastic. Incinerator attendants should receive proper instructions about
loading and temperature control. It should also be noted that the efficient operation of an incinerator
depends heavily on the right mix of materials in the waste being treated.
There are on-going concerns regarding the possible negative environmental effects of existing or
proposed incinerators, and efforts continue to make incinerators more environmentally friendly and
energy efficient.
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Disposal
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The disposal of laboratory and medical waste is subject to various regional, national and international
regulations and the latest versions of such relevant documents must be consulted before designing and
implementing a programme for handling, transportation and disposal of biohazardous waste. In general,
ash from incinerators may be handled as normal domestic waste and removed by local authorities.
Autoclaved waste may be disposed of by off-site incineration or in licensed landfill sites (see section on
Contaminated (infectious) materials for disposal in Chapter 3).
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Decontamination of prion-containing materials
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Prions, also referred to as unconventional infectious agents or agents of transmissible spongiform

encephalopathies, are believed to contain protein only. As mentioned previously, they can cause
Creutzfeldt-Jakob disease in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle, etc.
These infectious agents are unusually resistant to inactivation by most physical and chemical agents and
materials suspected of containing them require special processing before reuse or disposal.
To date, available data indicate that prions can be inactivated by a solution of 2 mol/l sodium
hydroxide (NaOH) containing 4.0 mol/l guanidinium hydrochloride (HNC(NH2)2.HCl) or guanidinium
isocyanate (HNC(NH2)2.HNCO) and sodium hypochlorite (NaOCl) (> 2% available chlorine) followed by
steam autoclaving at 132 C for 4.5 h.
Incineration is also an effective means of dealing with prion-contaminated materials (see section on
Precautions with materials that may contain unconventional agents in Chapter 10).
Summary
Proper use of chemical germicides and autoclaves will contribute to workplace safety while reducing the
risk from infectious agents. As far as possible, the number of germicidal chemicals to be used should be
limited, not only for economic reasons and better inventory control but also to avoid the loading of the
environment with potentially harmful chemicals.
There is much effort under way to make the chemical and physical means of inactivating
microorganisms in laboratories better and safer, with newer forms of sterilization and innovative
formulations of chemical germicides. At the same time, better methods for testing and validating the
claims of products sold for microbial decontamination are being developed. It is therefore important that
those designing laboratories and biosafety procedures to be used in them refer to the latest versions of
national/international standards and guidelines in this regard.
For further information see references 13 and 29-40.
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PART IV
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Chemical, fire and electrical safety
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15. Hazardous chemicals
Workers in microbiological laboratories are exposed to chemical hazards as well as to pathogenic

microorganisms. It is therefore vital that they have proper knowledge of the toxic effects of these
chemicals, the routes of exposure, and the hazards that may be associated with their handling and
storage. Material safety data sheets (MSDS), which describe the hazards associated with the use of a
given chemical, are available from the manufacturer, and should be made available in laboratories where
these chemicals are used, e.g. as part of a safety or operations manual.
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Definitions and classifications
Routes of exposure
Ingestion
Through broken skin
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Contact
Chemicals may cause irritation, sensitization, allergic reactions, respiratory

disease or cancer.
Contact with skin may cause chemical burns, conjunctivitis of the eyes, or
systemic poisoning.
Hazardous chemicals may be accidentally swallowed via mouth pipetting, or
contamination of food or drinks.
Hazardous chemicals may enter the body via cuts, abrasions or needle-sticks.
Storage of chemicals
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Inhalation
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Exposure to hazardous chemicals may occur in several ways:
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Hazardous chemicals are often defined and classified according to regulations written for the transport of
dangerous goods or by the hazards and degrees of danger they present. They may be listed by their
degree of reactivity, instability, fire or health hazard or by toxic effects.
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Only minimum amounts of the chemicals listed below should be stored in the laboratory for daily use.
Bulk stocks should be kept in specially designated rooms or buildings, which should have concrete floors
with sills at doorways to retain spills. Flammable substances should be stored separately in buildings that
are some distance from any others. To avoid ignition of flammable and explosive vapours by the sparking
of electrical contacts, light switches for these stores should be on the outside of the building and the lights
themselves should be in bulkheads.
Chemicals should not be stored in alphabetical order. Otherwise incompatible chemicals (see
below) may be in close proximity and some hazardous chemicals may be on high shelves. All large
bottles and all bottles containing strong acids and alkalis should be at floor level and in drip trays. Bottle
carriers and siphoning devices for filling bottles from bulk containers should be provided. Stepladders
should be provided where there are high shelves.
Incompatible chemicals
Many common laboratory chemicals react in a dangerous manner if they come into contact with one
another. Some such incompatible chemicals are listed below.
Acetic acid with chromic acid, nitric acid, hydroxyl compounds, ethylene glycol, perchloric acid,
peroxides and permanganates.
Acetone with concentrated sulfuric and nitric acid mixtures.
Acetylene with copper (tubing), halogens, silver, mercury and their compounds.
Alkali metals with water, carbon dioxide, carbon tetrachloride and other chlorinated hydrocarbons.
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Ammonia, anhydrous with mercury, halogens, calcium hypochlorite and hydrogen fluoride.
Ammonium nitrate with acids, metallic powders, flammable liquids, chlorates, nitrites, sulfur and finely
divided organic or combustible compounds.
Aniline with nitric acid and hydrogen peroxide.
Bromine with ammonia, acetylene, butadiene, butane, hydrogen, sodium carbide, turpentine and finely
divided metals
Carbon, activated with calcium hypochlorite and all oxidizing agents.
Chlorates with ammonium salts, acids, metal powders, sulfur and finely divided organic or combustible
compounds.
Chlorine with ammonia, acetylene, butadiene, benzene and other petroleum fractions, hydrogen,
sodium carbide, turpentine and finely divided metals.
Chlorine dioxide with ammonia, methane, phosphine and hydrogen sulfide.
Chromic acid with acetic acid, naphthalene, camphor, alcohol, glycerol, turpentine and other flammable
liquids.
Copper with acetylene, azides and hydrogen peroxide.
Cyanides with acids.
Flammable liquids with ammonium nitrate, chromic acid, hydrogen peroxide, nitric acid, sodium
peroxide and halogens.
Hydrocarbons with fluorine, chlorine, bromine, chromic acid and sodium peroxide.
Hydrogen peroxide with chromium, copper, iron, most other metals or their salts, flammable liquids and
other combustible products, aniline and nitromethane.
Hydrogen sulfide with fuming nitric acid and oxidizing gases.
Iodine with acetylene and ammonia.
Mercury with acetylene, fulminic acid and ammonia.
Nitric acid with acetic acid, chromic acid, hydrocyanic acid, aniline, carbon, hydrogen sulfide, fluids,
gases and other substances that are readily nitrated.
Oxygen with oils, greases, hydrogen and flammable liquids, solids and gases.
Oxalic acid with silver and mercury.
Perchloric acid with acetic anhydride, bismuth and its alloys, alcohol, paper, wood and other organic
materials.
Phosphorus pentoxide with water.
Potassium permanganate with glycerol, ethylene glycol, benzaldehyde and sulfuric acid
Silver with acetylene, oxalic acid, tartaric acid and ammonium compounds.
Sodium with carbon tetrachloride, carbon dioxide and water.
Sodium azide with lead, copper and other metals. This compound is commonly used as a preservative
but forms unstable, explosive compounds with metals. If it is flushed down sinks, the metal traps and
pipes may explode when worked on by a plumber.
Sodium peroxide with any oxidizable substance, e.g. methanol, glacial acetic acid, acetic anhydride,
benzaldehyde, carbon disulfide, glycerol, ethyl actetate and furfural
Sulfuric acid with chlorates, perchlorates, permanganates and water.
General rules
Substances in the left-hand column below should be stored and handled so that they cannot accidentally
come into contact with the corresponding substances in the right-hand column.
Alkali metals, e.g. sodium, potassium, caesium
and lithium
Carbon dioxide, chlorinated hydrocarbons, water
Halogens
Ammonia, acetylene, hydrocarbons
Acetic acid, hydrogen sulfide, aniline,

hydrocarbons, sulfuric acid
Oxidizing agents, e.g. chromic acid, nitric acid,

peroxides, permanganates
Toxic effects of chemicals

It is well known that some chemicals adversely affect the health of those who handle them or inhale their
vapours. Apart from overt poisons, a number of chemicals are known to have various toxic effects. The
respiratory system, blood, lungs, liver, kidneys and the gastrointestinal system, as well as other organs
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Hazardous chemicals
and tissues may be adversely affected or seriously damaged. Some chemicals are known to be
carcinogenic or teratogenic.
Some solvent vapours are toxic when inhaled. Apart from the more serious effects noted above,
exposure may result in impairments that show no immediate discernible effects on health, but can include
lack of coordination, drowsiness and similar symptoms, leading to an increased proneness to accidents.
Prolonged or repeated exposure to the liquid phase of many organic solvents can result in skin
damage. This may be due to a defatting effect, but allergic and corrosive symptoms may also arise.
Table 11 lists the reported adverse health effects of some common laboratory chemicals.
Table 11. Adverse health effects of some laboratory chemicals
Reported effects
Chemical
Chronic
Acetaldehyde (acetic aldehyde;

ethanal)
Eye and respiratory tract

irritation; narcosis
Bronchitis; liver damage
Acetic anhydride (acetyl oxide;

ethanoic anhydride)
Strong eye and upper respiratory

tract irritation; corrosive action
Acetone (dimethyl ketone;

2-propanone)
Slight eye, nose and throat

irritation; narcosis
Acetonitrile (methyl cyanide)
Respiratory irritation; cyanide

poisoning
Acrolein
Lacrimation; respiratory irritation
Ammonia
Eye irritation
Aniline (aminobenzene;
phenylamine)
Cyanosis due to
methaemoglobinaemia; slight
narcosis; respiratory paralysis
Benzene
Narcosis
Leukaemia; liver damage;

aplastic anaemia
Benzidine
Abdominal pain; nausea; skin

irritation
Carcinogenesis
Headache; nausea; slight

jaundice; loss of appetite;
narcosis
Liver and kidney damage;

gastrointestinal disturbances
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Carbon tetrachloride
(tetrachloromethane)
Pulmonary oedema
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Acute
Chloroform (trichloromethane)
Headache; nausea; slight

jaundice; loss of appetite;
narcosis
Cyanogen bromide
Abdominal pain; nausea;

diarrhoea; blurred vision
Pulmonary oedema
Cytochalasin
Mutagenesis
Diethyl ether
Vomiting; eye irritation
Addictive
Dioxane
Narcosis
Liver and kidney damage;

carcinogenesis
Formaldehyde (formalin)
Respiratory, skin and mucous

membrane irritation
Pulmonary oedema
Glutaral
Respiratory and mucous

membrane irritation
Mercury
Vomiting; diarrhoea; headache;
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Central nervous system

disturbance; swollen gums;
Chemical
Reported effects
Chronic
nausea; eye pain
loose teeth
Methanol (methyl alcohol)
Narcosis; mucous membrane

irritation
Damage to retina and optic

nerve
-Naphthylamine
Suspected carcinogen
-Naphthylamine
Carcinogenesis
Nitrobenzene (nitrobenzol)
Cyanosis due to
methaemoglobinaemia; slight
narcosis
Anaemia; reduced blood

pressure;
methaemoglobinaemia with
cyanosis; bladder irritation; liver
damage
Phenol
Abdominal pain; vomiting;

diarrhoea; skin irritation; eye
pain; corrosive action

disturbance; coma
Pyridine
Liver and kidney damage
Neurotoxicity
Selenium
Burning skin; eye pain; cough

disturbance; teratogenesis
Tetrahydrofuran (diethyl oxide;

tetramethyl oxide)
Narcosis; liver and kidney

damage; eye and respiratory
irritation
Thallium
Abdominal pain; vomiting;

nausea; diarrhoea
Neuropathy; visual problems;

muscle weakness; ataxia
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Narcosis
Trichloroethylene (ethinyl
trichloride)
Narcosis
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Toluene (methyl benzene;

phenyl methane; toluol)
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Carcinogenesis
Nonspecific neurological
impairment; addiction possible
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Liver damage; nonspecific

neurological impairment
Narcosis; headache; dizziness;

fatigue; nausea
impairment
o-Xylene (1,3-dimethylbenzene)

fatigue; nausea
impairment
p-Xylene (1,4-dimethylbenzene)

fatigue; nausea
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Explosive chemicals
Azides should not be allowed to come into contact with copper, e.g. in waste pipes and plumbing. Copper
azide will explode violently when subjected even to a mild impact.
Perchloric acid, if allowed to dry on woodwork, brickwork or fabric, will explode and cause a fire on
impact.
Picric acid and picrates are detonated by heat and impact.
Chemical spillage
Most manufacturers of laboratory chemicals issue charts describing methods for dealing with spillages.
Spillage charts and spillage kits are also available commercially. Appropriate charts should be displayed
in a prominent position in the laboratory. The following equipment should also be provided:
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Hazardous chemicals
protective clothing, e.g. heavy-duty rubber gloves; overshoes or rubber boots; respirators
scoops and dustpans
forceps for picking up broken glass
mops, cloths and paper towels
buckets
soda ash (sodium carbonate, Na2CO3) or sodium bicarbonate (NaHCO3) for neutralizing acids
sand
nonflammable detergent.
Spills should be neutralized as follows:
Acids and corrosive chemicals
With soda ash (sodium carbonate) or sodium bicarbonate
Alkalis
Cover with dry sand
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6.
Notify the biosafety officer and evacuate non-essential personnel from the area.
Attend to persons who may have been contaminated.
If the spilled material is flammable, extinguish all open flames, turn off gas in the room and adjacent
areas, and switch off electrical equipment that may spark.
Avoid breathing vapour from spilled material.
Establish exhaust ventilation if it is safe to do so.
Secure the necessary items (see above) to clean up the spillage.
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The following actions should be taken in the event of a spillage of a dangerous chemical.
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Compressed and liquefied gases
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In the event of a large chemical spillage the room should be evacuated and the windows opened, if
possible. If the material spilled is flammable, all open flames in the room concerned and those adjacent
should be extinguished and all electrical equipment that may spark switched off.
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Rooms where flammable gas cylinders are used should be identified by warning notices on the doors.
Not more than one cylinder of a flammable gas should be in the room at any one time. Spare cylinders
should be stored in another building at some distance from the laboratory. This store should be locked
and identified by a notice.
Compressed gas cylinders should be securely fixed (e.g. chained) to the wall or a solid bench so that
they are not dislodged by natural disasters.
Compressed gas cylinders and liquefied gas containers should not be placed near to radiators, open
flames or other heat sources, or sparking electrical equipment, or in direct sunlight.
The main high-pressure valve should be turned off when the equipment is not in use and when the
room is unoccupied.
Compressed gas cylinders must be transported with their caps in place and supported on trolleys.
Small, single-use gas cylinders must not be incinerated.
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16. Fire in the laboratory
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electrical overloading
poor electrical maintenance
excessively long gas tubing and electricity leads
equipment left switched on unnecessarily
naked flames
deteriorated gas tubing
misuse of matches
carelessness with flammable materials
flammable and explosive chemicals stored in ordinary refrigerators.
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Close cooperation between safety officers and local fire prevention officers is essential. Apart from
chemical hazards, the effects of fire on the possible dissemination of infectious material must be
considered. This may determine any burn out policies (i.e. consideration as to whether it is best to
extinguish or contain the fire).
The assistance of local fire prevention officers in the training of laboratory staff in fire prevention,
immediate action in case of fire, and the use of fire-fighting equipment is desirable.
Fire warnings, instructions and escape routes should be displayed prominently in each room and in
corridors and hallways.
Common causes of fires in laboratories are:
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Fire-fighting equipment should be placed near to the doors of rooms and at strategic points in
corridors and hallways (as advised by local fire prevention officers). This equipment should include
hoses, buckets (of water and sand), and the following types of fire extinguisher: water (H2O), carbon
dioxide (CO2), dry powder, and foam. The shelf-life of these extinguishers should be ascertained and
arrangements made for them to be regularly inspected and maintained. Their uses are shown in
Table 12.
Table 12. Types and uses of fire extinguishers
Do not use for
Use for
Water
Paper, wood fabric
Electrical fires, flammable liquids, burning

metals
CO2, powder
Flammable liquids and gases, electrical

fires
Alkali metals, paper
Dry powder
Flammable liquids and gases, alkali

metals, electrical fires
Foam
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Electrical fires
Water extinguishers are driven by carbon dioxide (CO2); care is necessary with CO2 powder extinguishers as the force of the jet
may spread burning materials; rooms should be well ventilated after use of BCF extinguishers.
For further information see reference 42.
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17. Electrical hazards
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Electric shock is life-threatening; electrical faults may cause fires. It is therefore essential that all electrical
installations and equipment are inspected and tested regularly, including earthing (grounding), and are
maintained by qualified electricians. Laboratory staff should not attempt to service any kind of electrical
equipment.
Voltages vary from country to country but even low voltages can be hazardous. Care should always
be taken to ensure that fuses of the correct rating are interposed between the equipment and the supply.
Circuit breakers and earth fault interrupters should be fitted into laboratory electrical circuits.
Note. Circuit breakers do not protect people; they are intended to protect wiring from overheating and
hence to prevent fires. Earth fault interrupters are intended to protect people from electric shock.
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wet or moist surfaces near to electrical equipment

long flexible electrical connecting cables
poor and perished insulation on cables
overloading of circuits by use of adapters
sparking equipment near to flammable substances and vapours
electrical equipment left switched on and unattended
use of the wrong type of fire extinguisher (water or foam instead of CO2) on electrical fires (see
Table 12).
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All laboratory electrical equipment should be earthed, preferably through three-prong plugs. Doubleinsulated devices, requiring only two-prong plugs, are rare in laboratories, but if present may require
separate earthing. An earth-free supply may become live as a result of an undetected fault.
All laboratory electrical equipment should conform to the national electrical safety standard or that of
the International Electrotechnical Commission.
Laboratory staff should be made aware of the following hazards:
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PART V
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Safety organization and training
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18. The biosafety officer and safety committee
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It is essential that each laboratory organization has a comprehensive safety policy, a safety or operations
manual, and a safety implementation programme. The responsibility for this normally rests with the
director or head of the institute or laboratory, who may, however, delegate certain duties to a biosafety
officer or other specialist officers.
It must be emphasized that laboratory safety is also the responsibility of all supervisors and
employees, and that individual workers are responsible for their own safety and that of their colleagues.
Employees are expected to perform their work safely and not place themselves or others at risk of injury
or illness due to unsafe or unhealthy conditions or actions. Laboratory workers should immediately report
unsafe acts or conditions to their supervisors.
Periodic safety audits by external independent consultants or specialists are desirable.
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Wherever possible a biosafety officer should be appointed to ensure that safety policies and programmes
are followed consistently throughout the laboratory. The biosafety officer executes these duties on behalf
of the head of the institute or laboratory. In small units, the biosafety officer may be a microbiologist or a
member of the technical staff, who may perform these duties on a defined part-time basis. Whatever the
degree of involvement in safety work, the person designated should have a sound microbiological
laboratory background, be actively involved in the work of the laboratory, and have experience and
training in the broader aspects of laboratory safety. He or she should not be an administrator or
technician involved in administrative or clerical activities.
The activities of the biosafety officer should include the following.
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1. Periodic internal safety audits on technical methods, chemicals, materials and equipment; these
audits should also include comprehensive workplace surveys to ensure compliance with appropriate
local and national safety and health policies and standards.
2. Discussions of infringements of the safety policy with the appropriate persons.
3. Verification that all members of the staff have received appropriate instruction and that they are
aware of all hazards, and that members of the medical, scientific and technical staff are competent to
handle infectious materials.
4. Provision of continuing instruction in safety for all personnel.
5. Provision of up-to-date safety literature and information to staff about changes in procedures,
technical methods, requirements, and the introduction of new equipment.
6. Investigation of all accidents and incidents involving the possible escape of potentially infected or
toxic material, even if there has been no personal injury or exposure, and reporting of the findings
and recommendations to the director and safety committee.
7. Giving assistance in following up illnesses or absences among laboratory staff where these absences
may be associated with the work and recorded as possible laboratory-acquired infections.
8. Ensuring that decontamination procedures are followed in the event of a spill or other incident
involving infectious material; a detailed, written record of such accidents and incidents should be kept
in case they may be related at a later date to a laboratory-acquired infection or other condition.
9. Ensuring that used materials are decontaminated, and that infectious wastes are safely disposed of
after treatment.
10. Ensuring the disinfection of any apparatus requiring repair or servicing before it is handled by nonlaboratory personnel.
11. Establishment of procedures for recording the receipt, movements and disposal of pathogenic
material and for notification by any research worker or laboratory of the introduction of infectious
materials that are new to the laboratory.
12. Advising the director of the presence of any agents that should be notified to the appropriate local or
national authorities and regulatory bodies.
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13. Reviewing the safety aspects of all plans, protocols and operating procedures for research work prior
to the implementation of these procedures.
14. Institution of a system of on call duties to deal with any emergencies that arise outside normal
working hours.
Safety committee
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If the institute is large enough, a safety committee should be constituted to recommend a safety policy
and to formulate a code of practice or safety or operations manual to serve as the basis of safety
practices in the individual laboratories, as advised by the biosafety officer. The safety committee should
also periodically review and update the safety policy as necessary.
Safety problems brought to the attention of the biosafety officer, along with information about how they
were dealt with, should be presented to the safety committee at regular meetings. Other functions of the
committee may include risk assessments of research plans, formulation of new safety policies, and
arbitration in disputes over safety matters.
The size and composition of the safety committee will depend on the size and nature of the laboratory,
on the work involved, and on the distribution of its work units or areas. The membership of the committee
should reflect the diverse occupational areas of the organization. In countries where there is national
legislation on health and safety, the composition of safety committees may already be determined. The
composition of a basic safety committee may be:
Elected by members
Biosafety officer
Medical adviser
Veterinarian
Representatives of professional staff
Representatives of technical staff
Representatives of management
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Chairperson
Members
General organization
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The safety committee may also include in its membership different departmental and specialist safety
officers (e.g. with expertise in radiation protection, industrial safety, fire prevention, etc.) and may at times
require advice from independent experts in various associated fields, the local authorities, and national
regulatory bodies. Community members may also be helpful if there is a particularly contentious or
sensitive protocol under discussion.
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The size and composition of a safety organization will depend on the requirements of the individual
laboratory and, in some cases, national regulations. Obviously, small, individual laboratories will not
require an elaborate organizational structure or full-time professional safety staff. In many instances,
where small individual institutions or laboratories are engaged in work with microorganisms in Risk
Groups 1 and 2, a safety organization can be developed for a group of them. Major biomedical
institutions may require a separate safety committee that confines its activities to specialized aspects of
its own programme.
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19. Safety rules for support staff
The safe and optimum operation of a laboratory is dependent to a great extent on the support staff, and it
is therefore essential that such personnel should be properly trained for their work.
As engineering, maintenance and cleaning personnel have to enter the premises and interact with the
laboratory staff, it is essential that they should perform their duties with due regard for appropriate safety
rules. They should apply locally adopted standard operational procedures and should be supervised.
Engineering and building maintenance services
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These services, which are concerned with the maintenance and repair of the structure, facilities and
equipment, have important support functions in the safety programme. For routine servicing and
maintenance it is not only convenient but also good safety policy to have available skilled engineers and
craftsmen who also have some knowledge of the nature of the work of the laboratory. Safety regulations
are better understood and observed by such staff; external engineers and others may be unaware of the
hazards and limitations that are placed upon them when they are working in a laboratory, and therefore
need much closer supervision by laboratory staff.
The testing of certain items of equipment after servicing is best carried out independently by or under
the supervision of the biosafety officer, e.g. testing the efficiency of biological safety cabinets after new
filters have been fitted, and of other equipment designed to minimize or retain aerosols.
Smaller laboratories or institutions that do not have internal engineering and maintenance services,
should establish, well in advance of any emergency, a good relationship with local engineers and builders
and familiarize them with the equipment and work of the laboratory.
Engineers and maintenance staff should not enter containment laboratories Biosafety Level 3 or
maximum containment laboratories Biosafety Level 4, except after clearance by, and under the
supervision of, the biosafety officer or the laboratory supervisor.
The staff of large facilities with several laboratories should receive training as outlined below.
Cleaning (domestic) services
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Laboratories should preferably be cleaned by staff who are under the control of the laboratory supervisor
and not by contract cleaners or staff responsible to other authorities. This practice fosters a safe,
cooperative working relationship between the laboratory and cleaning personnel. In particular it ensures
that cleaning staff will not be changed without warning.
In containment laboratories Biosafety Level 3 and maximum containment laboratories Biosafety
Level 4, the cleaning should preferably be done by the laboratory staff. Otherwise cleaning personnel
should work only after clearance by, and under the supervision of, the biosafety officer or the laboratory
supervisor.
The staff should receive training as outlined below.
The following rules are designed to aid in the prevention of laboratory-acquired infections in cleaning
personnel. Copies should be distributed to staff and posted in prominent positions.
Safety rules for domestic and cleaning staff

1. Always wear the protective clothing provided in the manner prescribed by the laboratory supervisor.
2. Take the protective clothing off when you leave a laboratory to visit another part of the building. Do
not wear protective clothing when visiting the staff room, the toilet or the canteen.
3. Wash your hands often, and always before leaving the laboratory or going to the toilet, or to the staff
room.
4. Do not eat or drink, or apply cosmetics in any laboratory. Use the staff room or the toilet.
5. Do not dust or clean any work benches without the permission of the laboratory staff.
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6. If you have an accident of any kind, or knock over or break any bottle, tube, jar or piece of equipment,
tell the biosafety officer or your supervisor or one of the laboratory staff at once.
7. Do not attempt to clear up after any accident without permission. Do not pick up broken glass with
your fingers. Use a dustpan and brush, or forceps. Follow the instructions of senior members of the
staff.
8. Do not enter any room that has a restricted entry sign on the door (e.g. the biohazard or radiation
hazard sign) unless authorized to do so.
9. Do not empty any discard containers in the laboratory unless a label or an instruction says that you
may do so.
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20. Training programmes
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A continuous, on-the-job safety training programme is essential to maintain safety awareness among the
laboratory and support staff. Laboratory supervisors, with the assistance of the biosafety officer and other
resource persons, play the key role in staff training. The effectiveness of biosafety training, indeed all
safety and health training, depends on management commitment, motivational factors, adequate initial
job training, good communications, and ultimately the organizations goals and objectives. The following
are critical elements for an effective biosafety training programme; in addition to taking account of these
elements it is recommended that the developers of any safety training programme become acquainted
with the principles of adult learning.
Needs assessment. This process includes defining the tasks involved, the order of importance (in
terms of frequency, criticality, complexity), and details of the steps necessary to accomplish them.
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Establishing training objectives. The needs assessment provides the information to establish the
objectives of the biosafety training programme. These are observable behaviours that the trainee is
expected to demonstrate, on the job, after the training. Objectives may acknowledge the conditions under
which certain activities or behaviours are performed and the required level of proficiency.
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Specifying training content and media. Content is the knowledge or skill that the trainee must
master to be able to meet the behavioural objectives. Those individuals who know the job and its
demands best usually define the content of the biosafety training programme. Other approaches used
may focus on the products of problem-solving exercises, or the design of learning measures to correct
mistakes people have made in using a skill. It is not clear that one teaching method (lectures, televised
instruction, computer-aided instruction, interactive video, etc.) is superior to another. Much depends on
specific training needs, the make-up of the trainee group, etc.
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Accounting for individual learning differences. Effective training must take into account the
characteristics or attributes of the trainees. Individuals and groups may differ in aptitude, literacy, culture,
spoken language and pre-training skill levels. How the training programme is viewed by trainees in terms
of improving their job performance or personal safety may dictate the approach used. Some individuals
are more visual or hands-on learners; others learn well from written materials. Any special needs of
employees must also be addressed, such as course adaptation for those with hearing impairments.
Specifying learning conditions. The instructional event (e.g. training course, videotape, written
materials, etc.) should not conflict with, inhibit or be unrelated to mastery of the skill or topic being taught.
For example, if the intent of the instruction is to develop capabilities in problem-solving techniques, the
instructional approach should stress thinking/reasoning approaches rather than rote memorization. The
instruction provided should require productive behaviour and/or appropriate feedback
(positive/accurate/credible). In addition, instructional events that provide opportunities for practice under
conditions similar to that of the job will enhance the transfer of the skill to the actual job.
Training evaluation. This provides information that helps to determine whether the instruction has
had the intended effect. Training evaluations generally take four forms:
measuring the trainees reaction to the instruction provided

measuring the trainees recollection and/or performance
assessing behavioural change on the job
measuring tangible results in terms of the organizations objectives or goals.
The most complete evaluation of a training effort involves assessments for each of the four areas. The
least efficient method of evaluation is to consider only the trainees reactions to the instruction as this
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may bear little relationship to the extent of actual learning. It should not be used as the sole measurement
of training effectiveness.
Training revision. Training evaluations rarely indicate that a training programme is a complete
success or failure because multiple criteria are used to measure results. Usually the data indicate a better
understanding, retention or application of some parts of the course material as compared with others.
Variation or gaps in knowledge or the desired competencies resulting from the training effort may reflect
the need to consider more training time, alternative instructional techniques, or more capable instructors.
A basic course on good laboratory practice that can be modified to suit the needs and objectives of
most laboratories is offered below. This is followed by five example training modules designed for
members of the laboratory and support staff. These technical modules can also be adapted locally, based
on the needs assessment and learning objectives of a particular laboratory or organization.
General
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1. Sources of laboratory infections

2. Laboratory hazards:
biological
chemical
physical, including fire and electrical hazards
3. Laboratory workers rights and duties in relation to safety measures
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Basic course: Good laboratory practice (GLP)
1. Access to laboratories
2. Personal hygiene
3. Protective clothing
Use of mechanical and other pipetting aids

Minimization of aerosol production
Proper use of biological safety cabinets
Proper use of autoclaves and sterilization equipment
Proper use of centrifuges
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Experimental procedures
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Preparatory procedures
Emergency procedures
1. First aid (in laboratories)
2. Spillages and breakages
3. Accidents
General laboratory maintenance

1.
2.
3.
4.
Storage of hazardous materials

Transport of hazardous materials
Handling and care of laboratory animals
Control of arthropods and rodents
Check-out procedures
1. Disposal of hazardous waste:
sterilization
incineration
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Training programmes
2. Decontamination procedures
3. Personal hygiene
Module 1 (the core module): Good microbiological technique (GMT)

This module is for scientists and technical staff who work in basic laboratories Biosafety Levels 1 and 2.
The course outlined below can be covered in one week.
As diagnostic laboratories cannot control the kind of specimens they receive and will almost certainly
on occasions be required to handle Risk Group 3 microorganisms, some training with these is also
necessary.
Course contents
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9.
10.
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12.
13.
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15.
16.
17.
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4.
5.
6.
7.
Classification of microorganisms according to Risk Groups on the basis of hazard; how this is
applied in different geographical areas
Laboratory infections; how they occur and the routes and modes of infection
Infections due to known accidents, e.g. accidental inoculation, spillage; prevention or minimization
Infections due to airborne infectious particles; how these particles (aerosols) are released
Measurement and control of aerosols; reduction of hazards by changing techniques and equipment
Protective clothing, face and eye protection, personal hygiene, immunization
Biological safety cabinets, Classes I and II only (Class III cabinets are unlikely to be used in these
laboratories)
Universal precautions (3) for handling blood and body fluids
Disposal of infected laboratory waste; principles and use of autoclaves and incinerators
Chemical disinfection: limitations and policies
The animal house: contamination control
Laboratory design: principles for a safe laboratory
Chemical and carcinogenic hazards; chemical fume cupboards; hazardous analytical equipment
Electrical and fire hazards
First aid for laboratory accidents
Emergency procedures
Mailing and shipping of infectious materials
Examination of existing codes of practice: formulation of local programmes; duties of biosafety
officers; sources of information
How to work without modern facilities
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Module 2: The safe laboratory environment

Module 2 is in two parts, one concerned with planning for safety and the other with organization for
safety. Both parts are aimed at senior scientific and technical laboratory staff, and at engineering,
architectural and administrative staff concerned with construction, maintenance and servicing of the
buildings. Discussion documents to be provided are relevant national biosafety guidelines and laboratory
planning and construction plans. Two days should be allowed for this course.
Course contents: Part 1

1. Size and distribution of rooms for different purposes; planning and building systems; furniture and
permanent equipment
2. Services: water, gas, electricity; alternative arrangements where no public services are available
3. Hygiene facilities: washing, toilets, etc.
4. Ventilation, including that of biological safety cabinets and fume cupboards
5. Waste disposal; contaminated and chemical waste; autoclaves and incinerators
6. Animal houses: planning, containment and control; exclusion of unwanted animals including
arthropods
7. Security against vandalism
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Course contents: Part 2

1.
2.
3.
4.
5.
Duties and functions of safety committees

Duties and functions of biosafety officers
Medical supervision; immunization programmes
Training of staff in outlying districts
Consideration of a detailed safety or operations manual and how it may be adapted to local
circumstances
6. Safety audits; how they are conducted and what auditors (inspectors) should look for
7. General safety services, e.g. fire precautions
8. Emergency-preparedness plans
Module 3: GLP for support staff
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This module, for a one-day course, is for the following groups of laboratory support staff who do not
normally have any laboratory training.
Group 1. Domestic staff who: clean the premises; dispose of contaminated and other laboratory
waste; wash and prepare glassware and other equipment; prepare and sterilize culture media and
reagents.
Group 2. Engineering and maintenance staff who: service laboratory facilities; repair equipment.
Group 3. Staff who: receive and sort pathological material brought to the laboratory; open mail; handle
request forms and laboratory records; pack infectious materials for mailing or shipping; drive vehicles that
carry infectious materials.
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Course contents
The nature of microorganisms and how they cause infections (all groups)
The work of the laboratory (all groups)
How to avoid infection in the laboratory; personal hygiene, protective clothing, eating and drinking;
biohazard signs and restricted areas (all groups)
4. Use and limitations of disinfectants (all groups)
5. Operation, control and testing of autoclaves, and incinerators (groups 1 and 2)
6. Hazards of particular equipment, e.g. biological safety cabinets, incubators, refrigerators,
homogenizers and centrifuges (group 2)
7. Hazards of internal transport and of receiving specimens. opening mail and handling records; how to
pack infectious materials for mailing and shipping; emergency action to be taken in case of accident
or spillage before the biosafety officer arrives (group 3)
8. Chemical, physical, mechanical, electrical and biological hazards (all groups)
9. The biosafety officer and his or her duties; explanation of the individual workers rights and
responsibilities according to national and local regulations; need to report accidents and unusual
occurrences; local security and fire precautions (all groups)
10. Simple first aid (all groups)
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2.
3.
Module 4: GLP for safety staff

This module is intended primarily for biosafety officers but other members of the safety committee should
be encouraged to attend. Five days should be allowed for this course.
Course contents
1. Outline of legal requirements for conduct in clinical and research laboratories; national occupational
health and safety legislation and examples from other, appropriate countries; responsibilities of
employers and employees; position of trade unions
2. Codes of practice and guidelines, safety or operations manuals; the employer's declarations of intent
and safety policies; implementation of safety programmes; duties of biosafety officers and safety
committees
3. Accident and incident reporting: mechanisms and channels in routine and emergency situations
4. Emergency programmes; preparing protocols for dealing with accidents, spillage, etc.
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Training programmes
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5. Medical surveillance: documentation of staff; immunization and sickness records; actions if laboratory
infection is suspected
6. Staff problems leading to aberrant behaviour and consequent hazards
7. Laboratory and animal house vandalism; security arrangements
8. Laboratory accidents: inoculation, spillage, breakage, equipment-related hazards: centrifuges,
homogenizers, pipetting, microbiological manipulations
9. Aerosols: how released (equipment and technique-related); hazards involved; measurement with slit
and cascade samplers: theory and practice of HEPA filtration
10. Supervision and instruction of staff in personal hygiene and use of protective clothing
11. Biological safety cabinets: classification, limitations and selective use; installation and testing
(biological challenge, chemical smokes); training of users
12. Design and testing of ventilation systems; graduated pressures; effluent control; clean air rooms
13. Principles and application of disinfection and sterilization; kinetics of bacterial destruction; autoclaves
and their control; use of thermocouples and indicators (chemical and biological)
14. Chemical and gaseous disinfectants; efficiency testing; disinfection policies; ultraviolet-irradiation
15. Packaging, mailing and shipping of infectious materials; national and international regulations;
emergency procedures
16. The animal house: containment and control; isolation from the environment
17. Hazardous chemicals: handling and storage; threshold limit values (or equivalent) and their
measurement
18. Radiation hazards: legal requirements and local control
19. Life support apparatus; lock-out procedures; the two-person rule
20. Fire precautions; burn-out decisions, i.e. whether to contain or extinguish the fire
21. Emergency preparedness plans
22. Emergency evacuation of staff and animals
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Module 5: GLP for specialist staff who handle microorganisms in Risk Groups 3
and 4
Course contents
Risk Groups 3 and 4 microorganisms: identity, associated diseases; clinical and epidemiological
features; natural reservoirs, vectors; modes of spread, prevention and treatment
2. Potential hazards of genetic manipulations and their relation to microorganisms in Risk Groups 3
and 4
3. Levels of containment for Risk Group 3 and 4 microorganisms
4. Classes I and II biological safety cabinets; containment laboratory Biosafety Level 3 procedures;
design of facilities for work with Risk Group 3 microorganisms
5. Class III biological safety cabinets; maximum containment laboratory Biosafety Level 4 procedures
for work with microorganisms in Risk Group 4
6. Specialized facilities for specific pathogens; monitoring of effluents: specialized clothing and hygiene
facilities
7. Specialized equipment: Class III biological safety cabinets, testing control, training in use; doubledoor pass-through autoclaves; warning against full dependence on mechanical equipment that may
give a false sense of security
8. Simple maintenance of electrical power, water, pressure equipment, etc. in areas where
maintenance staff cannot immediately be admitted
9. Medical supervision; immunization; emergency measures
10. Documentation of activities
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This module is for specialized scientific, technical and safety staff who will handle microorganisms in Risk
Groups 3 and 4. It is essential that safety personnel are trained up to the level of Module 4 before
following this module. Staff who have not received the proper training should not be given responsibility
for microorganisms in Risk Groups 3 and 4. The course lasts two to three days.
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PART VI
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Safety checklist
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21. Safety checklist
This checklist is intended to assist in assessments of the safety status of biomedical laboratories
Laboratory premises
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1. Do the premises meet national and local building requirements, including those relating to flood and
earthquake precautions if necessary?
2. Are the premises generally uncluttered and free from obstructions?
3. Are the premises clean?
4. Are there any structural defects in floors, stairways, walls and roofs?
5. Are floors and stairs uniform and slip-resistant?
6. Are there handrails on flights of stairs with more than four risers?
7. Are there guarded rails on floor openings?
8. Is the working space adequate for safe operation?
9. Are the circulation spaces and corridors adequate for the movement of people and large equipment?
10. Are the benches, furniture and fittings in good condition?
11. Are bench surfaces resistant to solvents and corrosive chemicals?
12. Is there a hand-wash basin in each laboratory room?
13. Are the premises constructed and maintained to prevent entry and harbourage of rodents and
arthropods?
14. Are all exposed steam and hot water pipes insulated or guarded to protect personnel?
15. Is an independent power support unit provided in case of power breakdown?
Storage facilities
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1. Are storage facilities, shelves, etc., arranged so that stores are secure against sliding, collapse or
falls?
2. Are storage facilities kept free from accumulations of rubbish, unwanted materials and objects that
present hazards from tripping, fire, explosion and harbourage of pests?
Sanitation and staff facilities

1. Are the premises maintained in a clean, orderly and sanitary condition?
2. Is drinking-water available?
3. Are clean and adequate toilet (WC) and washing facilities provided separately for male and female
staff?
4. Are hot and cold water, soap and towels provided?
5. Are separate changing rooms provided for male and female staff?
6. Is there accommodation (e.g. lockers) for street clothing for individual members of the staff?
7. Is there a staff room for lunch, etc.?
8. Are noise levels acceptable?
9. Is there an adequate organization for the collection and disposal of general household rubbish?
Heating and ventilation

1. Is there a comfortable working temperature?
2. Are blinds fitted to windows that are exposed to full sunlight?
3. Is the ventilation adequate, e.g. at least six changes of air per hour, especially in rooms that have
mechanical ventilation?
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4. Are there HEPA filters in the ventilation system?

5. Does mechanical ventilation compromise air flows in and around biological safety cabinets and fume
cupboards?
Lighting
1.
2.
3.
4.
5.
Is the general illumination adequate (e.g. 300400 lx)?

Is task (local) lighting provided at work benches?
Are there dark or ill-lit corners in rooms and corridors?
Are fluorescent lights parallel to the benches?
Are fluorescent lights colour-balanced?
Services
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1. Is each laboratory room provided with enough sinks, water, electricity and gas outlets for safe
working?
2. Is there an adequate inspection and maintenance programme for fuses, lights, cables, pipes, etc.?
3. Are faults corrected within a reasonable time?
4. Are internal engineering and maintenance services available, with skilled engineers and craftsmen
who also have some knowledge of the nature of the work of the laboratory?
5. If no internal engineering and maintenance services are available, have local engineers and builders
been contacted and familiarized with the equipment and work of the laboratory?
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1. Is the whole building securely locked when unoccupied?

2. Are doors and windows vandal-proof?
3. Are rooms containing hazardous materials and expensive equipment locked when unoccupied?
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Is there a fire alarm system?

Are the fire doors in good order?
Is the fire detection system in good working order and regularly tested?
Are fire alarm stations accessible?
Are all exits marked by proper, illuminated signs?
Is access to exits marked where the routes to them are not immediately visible?
Are all exits unobstructed and unlocked when the building is occupied?
Are any exits obscured by decorations, furniture or equipment?
Is access to exits arranged so that it is not necessary to pass through a high-hazard area to escape?
Do all exits lead to an open space?
Are corridors, aisles and circulation areas clear and unobstructed for movement of staff and firefighting equipment?
Is all fire-fighting equipment and apparatus easily identified by an appropriate colour code?
Are portable fire extinguishers maintained fully charged and in working order, and kept in designated
places at all times?
Are laboratory rooms with potential fire hazards equipped with appropriate extinguishers and/or fire
blankets for emergency use?
If flammable liquids and gases are used in any room, is the mechanical ventilation sufficient to
remove vapours before they reach a hazardous concentration?
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11.
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Fire prevention
Flammable liquid storage

1. Is the storage facility for bulk flammable liquids separated from the main building?
2. Is it clearly labelled as a fire-risk area?
3. Does it have a gravity or mechanical exhaust ventilation system that is separate from the main
building system?
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Safety checklist
4. Are the switches for lighting sealed or placed outside the building?
5. Are the light fittings inside sealed to protect against ignition of vapours by sparking?
6. Are flammable liquids stored in proper, ventilated containers that are made of non-combustible
materials?
7. Are the contents of all containers correctly described on the labels?
8. Are appropriate fire extinguishers and/or fire blankets placed outside but near to the flammable liquid
store?
9. Are No smoking signs clearly displayed inside and outside the flammable liquid store?
10. Are only minimum amounts of flammable substances stored in laboratory rooms?
11. Are they stored in properly constructed flammable storage cabinets?
12. Are these cabinets adequately labelled with Flammable liquid Fire hazard signs?
Electrical hazards
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1. Are all new electrical installations and all replacements, modifications or repairs made and
maintained in accordance with an electrical safety code?
2. Does the interior wiring have an earthed (grounded) conductor (i.e. a three-wire system)?
3. Are circuit breakers and earth fault interrupters fitted to all laboratory circuits?
4. Do all electrical appliances have testing laboratory approval?
5. Are the flexible connecting cables of all equipment as short as practicable, in good condition, and not
frayed, damaged or spliced?
6. Is each electric socket outlet used for only one appliance (no adapters to be used)?
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Compressed and liquefied gases
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Personal protection
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1. Is each portable gas container legibly marked with its contents and correctly colour-coded?
2. Are compressed-gas cylinders and their high-pressure and reduction valves regularly inspected for
safety?
3. Are reduction valves regularly maintained?
4. Is a pressure-relief device connected when a cylinder is in use?
5. Are protection caps in place when cylinders are not in use or are being transported?
6. Are all compressed gas cylinders secured so that they cannot fall, especially in the event of natural
disaster?
7. Are cylinders and liquid petroleum gas tanks kept away from sources of heat?
1. Is protective clothing of an approved design provided for all staff for normal work, e.g. gowns,
coveralls, aprons, gloves?
2. Is additional protective clothing provided for work with hazardous chemicals and radioactive and
carcinogenic substances e.g. rubber aprons and gloves for chemicals and for dealing with spillages;
heat-resistant gloves for unloading autoclaves and ovens.
3. Are safety glasses, goggles and shields (visors) provided?
4. Are there eye-wash stations?
5. Are there emergency showers (drench facilities)?
6. Is radiation protection in accordance with national and international standards, including provision of
dosimeters?
7. Are masks available?
8. Are respirators available, regularly cleaned, disinfected, inspected and stored in a clean and sanitary
condition?
9. Are appropriate filters provided for the correct types of respirators, e.g. HEPA filters for
microorganisms, appropriate filters for gases or particulates?
10. Are respirators fit-tested?
Health and safety of staff

1. Is there an occupational health service?
2. Are first-aid boxes provided at strategic places?
91
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3. Are qualified first-aiders available?

4. Are such first-aiders trained to deal with emergencies peculiar to the laboratory, e.g. contact with
corrosive chemicals, accidental ingestion of poisons and infectious materials?
5. Are non-laboratory workers, e.g. domestic and clerical staff, instructed on the potential hazards of the
laboratory and the material it handles?
6. Are notices prominently posted giving clear information about the location of first-aiders, telephone
numbers of emergency services, etc.?
7. Are women of childbearing age warned of the consequences of work with certain microorganisms,
carcinogens, mutagens and teratogens?
8. Are women of childbearing age told that if they are, or suspect that they are, pregnant they should
inform the appropriate member of the medical/scientific staff so that alternative working arrangements
may be made for them if necessary?
9. Is there an immunization programme relevant to the work of the laboratory?
10. Are skin tests and/or radiological facilities available for staff who work with tuberculous materials or
other materials requiring such measures?
11. Are proper records maintained of illnesses and accidents?
12. Are warning and accident prevention signs used to minimize work hazards?
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Is all equipment certified safe for use?

Are procedures available for decontaminating equipment prior to maintenance?
Are biological safety cabinets and fume cupboards regularly tested and serviced?
Are autoclaves and other pressure vessels regularly inspected?
Are centrifuge buckets and rotors regularly inspected?
Are pipettes used instead of hypodermic needles?
Is cracked and chipped glassware always discarded and not reused?
Are there safe receptacles for broken glass?
Are plastics used instead of glass where feasible?
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1.
2.
3.
4.
5.
6.
7.
8.
9.
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Infectious materials
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1. Are specimens received in a safe condition?

2. Are records kept of incoming materials?
3. Are specimens unpacked in biological safety cabinets with care and attention to possible breakage
and leakage?
4. Are gloves worn for unpacking specimens?
5. Are work benches kept clean and tidy?
6. Do discarded infectious materials, e.g. cultures, accumulate on benches and other places?
7. Are discarded infectious materials removed daily or more often and disposed of safely?
8. Are all members of the staff aware of procedures for dealing with breakage and spillage of cultures
and infectious materials?
9. Is the performance of sterilizers checked by chemical, physical and biological indicators?
10. Are centrifuges decontaminated daily?
11. Are sealed buckets provided for centrifuges?
12. Are appropriate disinfectants used correctly?
13. Is there special training for staff who work in containment laboratories Biosafety Level 3 and
maximum containment laboratories Biosafety Level 4?
Chemicals and radioactive substances

1.
2.
3.
4.
5.
6.
7.
Are incompatible chemicals effectively separated when stored or handled?

Are all chemicals correctly labelled with names and warnings?
Are chemical hazard warning charts prominently displayed?
Are spillage clearance kits provided?
Are staff trained to deal with spillages?
Are flammable substances correctly and safely stored in minimal amounts in approved cabinets?
Are bottle carriers provided?
92
Safety checklist
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8. Is a radiation protection officer or reference manual available for consultation?

9. Are proper records maintained of stocks and use of radioactive substances?
10. Are radioactivity screens and bench--counters provided?
93
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Washington, DC, U.S. Department of Health and Human Services/Centers for Disease Control and
Prevention/National Institutes of Health, 1999.
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Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the
protection of workers from risks related to exposure to biological agents at work. Official Journal,
2000, L 262:21-45.
3.
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European Council. Council Directive 98/81/EC of 26 October 1998 amending Directive 90/219/EEC
on the contained use of genetically modified microorganisms. Official Journal, 1998, L330:13-31.
6.
Hunt GJ, Tabachnick WJ. Handling small arbovirus vectors safely during biosafety level 3
containment: Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) and exotic bluetongue
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The Subcommittee on arthropod-borne viruses. Laboratory safety for arboviruses and certain other
viruses of vertebrates. American Journal of Tropical and Medical Hygiene, 1980, 29:1357-1381.
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National Research Council. Occupational health and safety in the care and use of research animals.
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Richmond JY, Quimby F. Considerations for working safely with infectious disease agents in
research animals. In: Zak O, Sande MA, eds. Handbook of animal models of infection. London,
Academic Press, 1999:69-74.
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10. National Sanitation Foundation. Class II (laminar flow) biohazard cabinetry. Ann Arbor, MI, 2002
(NSF/ANSI 49-2002).
11. Richmond JY, McKinney RW. Primary containment for biohazards: selection, installation and use of
biological safety cabinets, 2nd ed. Washington, DC, U.S. Department of Health and Human
Services/Centers for Disease Control and Prevention/National Institutes of Health, 2000.
12. Collins CH, Kennedy DA. Laboratory acquired infections: history, incidence, causes and prevention,
4th ed. Oxford, Butterworth-Heinemann, 1999.
13. Health Canada. Laboratory biosafety manual, 2nd ed. Ottawa, Minister of Supply and Services
Canada, 1996.
14. British Standards Institution. Microbiological safety cabinets. Recommendations for information to be
exchanged between purchaser, vendor and installer and recommendations for installation. London,
1992 (Standard BS 5726-2:1992).
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and maintenance. London, 1992 (Standard BS 5726-4:1992).
16. Canadian Standards Association. Biological containment cabinets (Class I and II): installation and
field testing. Toronto, 1995 (Standard Z316.3-95 (R2000)).
17. Standards Australia. Biological safety cabinets - biological safety cabinets (Class I) for personnel
and environment protection. Sydney, Standards Australia International, 1994 (Standard AS 2252.11994).
18. Standards Australia. Biological safety cabinets - laminar flow biological safety cabinets (Class II) for
personnel, environment and product protection. Sydney, Standards Australia International, 1994
(Standard AS 2252.2-1994).
19. Standards Australia/ Standards New Zealand. Biological safety cabinets - installation and use.
Sydney, Standards Australia International, 2000 (Standard AS/NZS 2647:2000).
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20. Standards Australia/ Standards New Zealand. Safety in laboratories - microbiological aspects and
containment facilities. Sydney, Standards Australia International, 2002 (Standard AS/NZS
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21. Advisory Committee on Dangerous Pathogens. Guidance on the use, testing and maintenance of
laboratory and animal flexible film isolators. London, Health and Safety Executive, 1990.
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22. Safar J et al. Prions. In: Richmond JY, McKinney RW, eds. Biosafety in microbiological and
biomedical laboratories, 4th ed. Washington, DC, U.S. Department of Health and Human Services,
1999:134-143.
23. Bellinger-Kawahara C et al. Purified scrapie prions resist inactivation by UV irradiation. Journal of
Virology , 1987, 61:159-166.
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24. Berg P et al. Asilomar conference on recombinant DNA molecules. Science, 1975, 188:991-994.
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25. Toloza EM et al. In vivo cancer gene therapy with a recombinant interleukin-2 adenovirus vector.
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26. O'Malley BW Jr et al. Limitations of adenovirus-mediated interleukin-2 gene therapy for oral cancer.
Laryngoscope, 1999, 109:389-395.
27. World Health Organization. Maintenance and distribution of transgenic mice susceptible to human
viruses. Memorandum from a WHO meeting. Bulletin of the World Health Organization, 1993,
71:497-502.
28. Health Services Advisory Committee. Safe working and the prevention of infection in clinical
laboratories. London, HSE Books, 1991.
29. Ascenzi JM. Handbook of disinfectants and antiseptics. New York, NY, Marcel Dekker, 1996.
30. Block SS. Disinfection, sterilization & preservation, 5th ed. Philadelphia, PA, Lippincott Williams &
Wilkins, 2001.
31. Russell AD, Hugo WB, Ayliffe GAJ. Disinfection, preservation and sterilization, 3rd ed. Oxford,
Blackwell Scientific, 1999.
32. Rutala WA. APIC guideline for selection and use of disinfectants. American Journal of Infection
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33. Sattar SA, Springthorpe VS, Rochon M. A product based on accelerated and stabilized hydrogen
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34. Schneider PM. Emerging low temperature sterilization technologies. In: Rutala WA, eds. Disinfection
& sterilization in health care. Champlain, NY, Polyscience, 1997:79-92.
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care. Champlain, NY, Polyscience, 1997:273-280.
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37. Taylor DM. Transmissible degenerative encephalopathies: inactivation of the unconventional causal
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39. Springthorpe VS, Sattar SA. Chemical disinfection of virus-contaminated surfaces. CRC Critical
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40. Sattar SA et al. The inactivation of HIV: an update. Reviews in Medical Microbiology, 1994, 5:139150.
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41. Eggimann W, Bastian C. Safety in handling chemical substances at a global level. Immunological
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42. Furr AK. CRC handbook of laboratory safety, 5th ed. Boca Raton, FL, CRC Press LLC, 2000.
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43. Lenga RE. The Sigma-Aldrich Library of Chemical Safety Data, 2nd ed. Milwaukee, WI, Aldrich
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47. International Electrotechnical Commission. Medical electrical equipment - Part 1-1: General
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Geneva, 2000 (IEC 60601-1-1 (2000-12)).
48. International Electrotechnical Commission. Medical electrical equipment - Part 1-2: General
requirements for safety - Collateral standard: Electromagnetic compatibility - Requirements and
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49. Cohen A and Colligan MJ. Assessing occupational safety and health training. Cincinnati, OH,
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Human performance and productivity: stress and performance effectiveness, Vol. 3. Hillsdale, NJ,
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51. Kroehnert G. Basic training for trainers: a handbook for new trainers. Sidney, McGraw-Hill, 1995.
97
ANNEX 1
Immunization of staff
It is recommended that all laboratory personnel receive protective immunization against the following
1
diseases: diphtheria, hepatitis B, measles, mumps, poliomyelitis, rubella, tetanus, tuberculosis ,
typhoid fever. Some workers may have been immunized during childhood but documentary evidence
of current protection should be obtained.
Note. BCG does not appear to give as much protection against tuberculosis in some parts of the
world, e.g. the Indian subcontinent, as in others; see BCG vaccination policies: report of a WHO
Study Group. Geneva, World Health Organization, 1980 (WHO Technical Report Series, No. 652),
and Fine PE et al. Issues relating to the use of BCG in immunization programmes. A discussion
2
document. Geneva, World Health Organization, 1999 (unpublished document WHO/V&B/99.23) .
2.
All persons who work with or who handle animals infected with the following agents should be given
the appropriate vaccine or toxoid, and facilities for specific clinical case management following
accidental infections should be available: Bacillus anthracis, Clostridium botulinum, Francisella
tularensis type A, Haemophilus influenzae, Japanese B encephalitis virus, Mycobacterium leprae,
3
Neisseria meningitidis, Yersinia pestis, hepatitis A virus, influenza virus, louping ill virus , rabies
3
virus, Rift Valley fever virus, tick-borne encephalitis viruses (Absettarov virus, Hanzalova virus and
Omsk haemorrhagic fever virus), varicella-zoster virus, Venezuelan equine encephalomyelitis virus,
yellow fever virus. Vaccinia vaccine is recommended for persons who work with orthopoxviruses.
Other vaccines are available and may be indicated in specific circumstances for laboratory workers
at high risk.
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1.
Caused by Mycobacterium tuberculosis, M. bovis and M. africanum.

Obtainable on request from Department of Vaccines and Other Biologicals, World Health Organization, 1211 Geneva 27,
Switzerland (http://www.who.int/gpv-documents/).
3
These viruses are antigenically very similar; immunization against one is expected to give protection against others.
2
98
ANNEX 2
WHO Biosafety Collaborating Centres
Information on the availability of training courses, aids and materials may be obtained by writing to any of
the following:
Biosafety Programme, Department of Communicable Disease Surveillance and Response, CSR
Office in Lyon, World Health Organization, 58 Avenue Debourg, 69007 Lyon, France.
http://www.who.int/emc/
WHO Collaborating Centre for Biological Safety, Swedish Institute for Infectious Disease Control,
Nobels Vg 18, S-171 82 Solna, Sweden. http://www.smittskyddsinstitutet.se/English/english.htm
WHO Collaborating Centre on Biosafety Technology and Consultative Services, Office of Biosafety,
Health Canada, Tunneys Pasture 0700A1, Ottawa, Ontario, Canada K1A 0L2.
http://www.hc-sc.gc.ca/hpb/lcdc/biosafty
WHO Collaborating Centre for Applied Biosafety Programmes and Training, Office of Health and
Safety, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop F05, Atlanta, GA
30333, USA. http://www.cdc.gov/
WHO Collaborating Centre for Applied Biosafety Programmes and Research, Occupational Safety
and Health Branch, Division of Safety, National Institutes of Health, 13/3K04 13 South Drive
MSC 5760, Bethesda, MD 20892-5760, USA. http://www.nih.gov/
WHO Collaborating Centre for Biosafety, Victorian Infectious Diseases Reference Laboratory, 10
Wreckyn St, Nth Melbourne, Victoria 3051, Australia. Postal address: Locked Bag 815, PO Carlton
Sth, Victoria 3053, Australia. http://www.vidrl.org.au/
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99

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in

SECOND EDITION (REVISED)

WORLD HEALTH ORGANIZATION

Basic laboratories Biosafety Levels 1 and 2............................................................................... 10

The containment laboratory Biosafety Level 3 ........................................................................... 17

The maximum containment laboratory Biosafety Level 4 ......................................................... 20

Laboratory animal facilities ............................................................................................................. 22

PART II Laboratory equipment

Biological safety cabinets................................................................................................................ 29

Laboratory Biosafety Manual, 2nd revised edition

Equipment designed to reduce biological hazards....................................................................... 39

10. Safe laboratory techniques.............................................................................................................. 45

11. Biosafety and recombinant DNA technology................................................................................. 52

12. Transport of infectious substances................................................................................................ 55

13. Contingency plans and emergency procedures............................................................................ 56

General rules ................................................................................................................................... 70

19. Safety rules for support staff........................................................................................................... 79

20. Training programmes ....................................................................................................................... 81

21. Safety checklist ................................................................................................................................. 87

W. Emmett Barkley, PhD

Dr Ingegerd Kallings, Swedish Institute of Infectious Disease Control, Stockholm, Sweden

Risk Group 2 (moderate individual risk, low community risk)

A microorganism that is unlikely to cause human or animal disease.

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

Table 1. Classification of infective microorganisms by risk group

Table 2. Relation of risk groups to biosafety levels, practices and equipment

None; open bench

GMT plus protective

Open bench plus

Laboratory Biosafety Manual, 2nd revised edition

As Level 2 plus special

BSC and/or other

As Level 3 plus airlock

Class III BSC, or

BSC, biological safety cabinet; GMT, good microbiological technique

Pathogenicity of the organism.

Countries (regions) should draw up a national (regional) classification of microorganisms, by risk

Table 3. Summary of biosafety level requirements

Room sealable for decontamination

inward air flow

mechanical via building system

HEPA filtered air exhaust

Airlock with shower

Anteroom with shower

Biological safety cabinets:

pathogenicity of the agent and infectious dose

Specimens for which there is limited information

medical data on the patient

Laboratory Biosafety Manual, 2nd revised edition

Risk assessment and genetically modified microorganisms

Hazards associated with the host/recipient

susceptibility of the host

Hazards arising directly from the inserted gene

Hazards arising from the alteration of existing pathogenic traits

Is there an increase in infectivity or pathogenicity?

For further information see Chapter 11, and references 4 and 5.