Biosafety Laboratory Manual Biosafety7

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World Health Organization

Geneva

2004

Laboratory biosafety manual

Third edition

WHO Library Cataloguing-in-Publication Data

World Health Organization.

Laboratory biosafety manual. – 3rd ed.

1.Containment of biohazards - methods 2.Laboratories - standards 3.Laboratory

infection - prevention and control 4.Manuals I.Title.

ISBN 92 4 154650 6 (LC/NLM classification: QY 25) WHO/CDS/CSR/LYO/2004.11

Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22

791 2476; fax: +41 22 791 4857; email: bookorders@who.int). Requests for permission to reproduce or

translate WHO publications – whether for sale or for noncommercial distribution – should be addressed

to Publications, at the above address (fax: +41 22 791 4806; email: permissions@who.int).

The designations employed and the presentation of the material in this publication do not imply the

expression of any opinion whatsoever on the part 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 the World Health Organization 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.

The World Health Organization does not warrant that the information contained in this publication is

complete and correct and shall not be liable for any damages incurred as a result of its use.

Printed in Malta

This publication was supported by Grant/Cooperative Agreement Number U50/CCU012445-08

from the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. Its contents are

solely the responsibility of the authors and do not necessarily represent the official views of the

CDC.

Contents

• iii •

Foreword vii

Acknowledgements viii

1. General principles 1

Introduction 1

PART I. Biosafety guidelines 5

2. Microbiological risk assessment 7

Specimens for which there is limited information 8

Risk assessment and genetically modified microorganisms 8

3. Basic laboratories – Biosafety Levels 1 and 2 9

Code of practice 9

Laboratory design and facilities 12

Laboratory equipment 14

Health and medical surveillance 16

Training 16

Waste handling 17

Chemical, fire, electrical, radiation and equipment safety 19

4. The containment laboratory – Biosafety Level 3 20

Code of practice 20

Laboratory design and facilities 21

Laboratory equipment 22

Health and medical surveillance 22

5. The maximum containment laboratory – Biosafety Level 4 25

Code of practice 25

Laboratory design and facilities 25

6. Laboratory animal facilities 28

Animal facility – Biosafety Level 1 29

Animal facility – Biosafety Level 2 29

Animal facility – Biosafety Level 3 30

Animal facility – Biosafety Level 4 31

Invertebrates 32

7. Guidelines for laboratory/facility commissioning 33

8. Guidelines for laboratory/facility certification 36

PART II. Laboratory biosecurity 45

9. Laboratory biosecurity concepts 47

PART III. Laboratory equipment 49

10. Biological safety cabinets 51

Class I biological safety cabinet 51

Class II biological safety cabinets 53

Class III biological safety cabinet 56

Biological safety cabinet air connections 56

Selection of a biological safety cabinet 57

Using biological safety cabinets in the laboratory 57

11. Safety equipment 61

Negative-pressure flexible-film isolators 61

Pipetting aids 63

Homogenizers, shakers, blenders and sonicators 63

Disposable transfer loops 64

Microincinerators 64

Personal protective equipment and clothing 64

PART IV. Good microbiological techniques 67

12. Laboratory techniques 69

Safe handling of specimens in the laboratory 69

Use of pipettes and pipetting aids 70

Avoiding the dispersal of infectious materials 70

Use of biological safety cabinets 70

Avoiding ingestion of infectious materials and contact with skin and eyes 71

Avoiding injection of infectious materials 71

Separation of serum 72

Use of centrifuges 72

Use of homogenizers, shakers, blenders and sonicators 73

Use of tissue grinders 73

Care and use of refrigerators and freezers 73

Opening of ampoules containing lyophilized infectious materials 74

Storage of ampoules containing infectious materials 74

Standard precautions with blood and other body fluids, tissues and excreta 74

Precautions with materials that may contain prions 76

13. Contingency plans and emergency procedures 78

Contingency plan 78

Emergency procedures for microbiological laboratories 79

14. Disinfection and sterilization 82

Definitions 82

Cleaning laboratory materials 83

• iv •

LABORATORY BIOSAFETY MANUAL

Chemical germicides 83

Local environmental decontamination 88

Decontamination of biological safety cabinets 89

Hand-washing/hand decontamination 90

Heat disinfection and sterilization 90

Incineration 92

Disposal 93

15. Introduction to the transport of infectious substances 94

International transport regulations 94

The basic triple packaging system 95

Spill clean-up procedure 95

PART V. Introduction to biotechnology 99

16. Biosafety and recombinant DNA technology 101

Biosafety considerations for biological expression systems 102

Biosafety considerations for expression vectors 102

Viral vectors for gene transfer 102

Transgenic and “knock-out” animals 102

Transgenic plants 103

Risk assessments for genetically modified organisms 103

Further considerations 104

PART VI. Chemical, fire and electrical safety 105

17. Hazardous chemicals 107

Routes of exposure 107

Storage of chemicals 107

General rules regarding chemical incompatibilities 107

Toxic effects of chemicals 107

Explosive chemicals 108

Chemical spills 108

Compressed and liquefied gases 109

18. Additional laboratory hazards 110

Fire hazards 110

Electrical hazards 111

Noise 111

Ionizing radiation 111

PART VII. Safety organization and training 115

19. The biosafety officer and biosafety committee 117

Biosafety officer 117

Biosafety committee 118

• v •

CONTENTS

20. Safety for support staff 119

Engineering and building maintenance services 119

Cleaning (domestic) services 119

21. Training programmes 120

PART VIII. Safety checklist 123

22. Safety checklist 125

Laboratory premises 125

Storage facilities 125

Sanitation and staff facilities 126

Heating and ventilation 126

Lighting 126

Services 126

Laboratory biosecurity 127

Fire prevention and fire protection 127

Flammable liquid storage 128

Compressed and liquefied gases 128

Electrical hazards 128

Personal protection 129

Health and safety of staff 129

Laboratory equipment 130

Infectious materials 130

Chemicals and radioactive substances 130

PART IX. References, annexes and index 133

References 135

Annex 1 First aid 138

Annex 2 Immunization of staff 139

Annex 3 WHO Biosafety Collaborating Centres 140

Annex 4 Equipment safety 141

Equipment that may create a hazard 141

Annex 5 Chemicals: hazards and precautions 145

Index 170

• vi •

LABORATORY BIOSAFETY MANUAL

Foreword

• vii •

The World Health Organization (WHO) has long recognized that safety and, in

particular, biological safety are important international issues. WHO published the

first edition of the Laboratory biosafety manual in 1983. The manual encouraged

countries to accept and implement basic concepts in biological safety and to develop

national codes of practice for the safe handling of pathogenic microorganisms in

laboratories within their geographical borders. Since 1983, many countries have used

the expert guidance provided in the manual to develop such codes of practice. A second

edition of the manual was published in 1993.

WHO continues to provide international leadership in biosafety through this third

edition of the manual by addressing biological safety and security issues facing us in

the current millennium. The third edition stresses throughout the importance of

personal responsibility. New chapters have been added on risk assessment, safe use of

recombinant DNA technology and transport of infectious materials. Recent world

events have revealed new threats to public health through deliberate misuse and release

of microbiological agents and toxins. The third edition therefore also introduces

biosecurity concepts – the protection of microbiological assets from theft, loss or

diversion, which could lead to the inappropriate use of these agents to cause public

health harm. This edition also includes safety information from the 1997 WHO

publication Safety in health-care laboratories (1).

The third edition of the WHO Laboratory biosafety manual is a helpful reference

and guide to nations that accept the challenge to develop and establish national codes

of practice for securing microbiological assets, yet ensuring their availability for clinical,

research and epidemiological purposes.

Dr A. Asamoa-Baah

Assistant Director-General

Communicable Diseases

World Health Organization

Geneva, Switzerland

Acknowledgements

• viii •

The development of this third edition of the Laboratory biosafety manual has been

made possible through the contributions of the following, whose expertise is gratefully

acknowledged:

Dr W. Emmett Barkley, Howard Hughes Medical Institute, Chevy Chase, MD, USA

Dr Murray L. Cohen, Centers for Disease Control and Prevention, Atlanta, GA, USA

(retired)

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

Sweden

Ms Mary Ellen Kennedy, Consultant in Biosafety, Ashton, Ontario, Canada

Ms Margery Kennett, Victorian Infectious Diseases Reference Laboratory, North Mel-

bourne, Australia (retired)

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 (retired)

Dr Syed A. Sattar, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada

Dr Deborah E. Wilson, Division of Occupational Health and Safety, Office of Research

Services, National Institutes of Health, Department of Health and Human Serv-

ices, Washington, DC, USA

Dr Riccardo Wittek, Institute of Animal Biology, University of Lausanne, Lausanne,

Switzerland

The assistance of the following is also gratefully acknowledged:

Ms Maureen Best, Office of Laboratory Security, Health Canada, Ottawa, Canada

Dr Mike Catton, Victorian Infectious Diseases Reference Laboratory, North Melbourne,

Australia

Dr Shanna Nesby, Office of Health and Safety, Centers for Disease Control and Pre-

vention, Atlanta, GA, USA

Dr Stefan Wagener, Canadian Science Centre for Human and Animal Health, Winni-

peg, Canada

The writers and reviewers also wish to acknowledge the original contributions of the

many professionals whose work was embodied in the first and second editions of the

Laboratory biosafety manual and in the 1997 WHO publication Safety in health-care

laboratories (1).

• 1 •

1. General principles

Introduction

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. Table 1 describes the risk groups.

Table 1. Classification of infective microorganisms by risk group

Risk Group 1

(no or low individual and community risk)

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

Risk Group 2

(moderate individual risk, low community risk)

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)

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.

Laboratory facilities are designated as basic – Biosafety Level 1, basic – Biosafety Level 2,

containment – Biosafety Level 3, and maximum containment – Biosafety Level 4.

Biosafety level designations are based on a composite of the design features,

construction, containment facilities, equipment, practices and operational procedures

required for working with agents from the various risk groups. Table 2 relates but

does not “equate” risk groups to the biosafety level of laboratories designed to work

with organisms in each risk group.

Countries (regions) should draw up a national (regional) classification of

microorganisms, by risk group, taking into account:

• 2 •

LABORATORY BIOSAFETY MANUAL

1. Pathogenicity of the organism.

2. 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 environ-

mental hygiene.

3. 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.

4. Local availability of effective treatment. This includes passive immunization,

postexposure vaccination and use of antimicrobials, antivirals and chemo-

therapeutic agents, and should take into consideration the possibility of the

emergence of drug-resistant strains.

The assignment of an agent to a biosafety level for laboratory work must be based on

a risk assessment. Such an assessment will take the risk group as well as other factors

into consideration in establishing the appropriate biosafety level. For example, an agent

that is assigned to Risk Group 2 may generally require Biosafety Level 2 facilities,

equipment, practices and procedures for safe conduct of work. However, if particular

experiments require the generation of high-concentration aerosols, then Biosafety

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

RISK BIOSAFETY LABORATORY LABORATORY SAFETY

GROUP LEVEL TYPE PRACTICES EQUIPMENT

1Basic – Basic teaching, GMT None; open bench

Biosafety research work

Level 1

2Basic – Primary health GMT plus protective Open bench plus BSC

Biosafety services; diagnostic clothing, biohazard for potential aerosols

Level 2 services, research sign

3Containment – Special diagnostic As Level 2 plus BSC and/or other

Biosafety services, research special clothing, primary devices for all

Level 3 controlled access, activities

directional airflow

4Maximum Dangerous pathogen As Level 3 plus Class III BSC, or

containment – units airlock entry, shower positive pressure suits

Biosafety exit, special waste in conjunction with

Level 4 disposal Class II BSCs, double-

ended autoclave

(through the wall),

filtered air

BSC, biological safety cabinet; GMT, good microbiological techniques (see Part IV of this manual)

• 3 •

Level 3 may be more appropriate to provide the necessary degree of safety, since it

ensures superior containment of aerosols in the laboratory workplace. The biosafety

level assigned for the specific work to be done is therefore driven by professional

judgement based on a risk assessment, rather than by automatic assignment of a

laboratory biosafety level according to the particular risk group designation of the

pathogenic agent to be used (see Chapter 2).

Table 3 summarizes the facility requirements at the four biosafety levels.

Table 3. Summary of biosafety level requirements

BIOSAFETY LEVEL

1234

Isolationa of laboratory No No Yes Yes

Room sealable for decontamination No No Yes Yes

Ventilation:

—inward airflow No Desirable Yes Yes

—controlled ventilating system No Desirable Yes Yes

—HEPA-filtered air exhaust No No Yes/NobYes

Double-door entry No No Yes Yes

Airlock No No No Yes

Airlock with shower No No No Yes

Anteroom No No Yes —

Anteroom with shower No No Yes/NocNo

Effluent treatment No No Yes/NocYes

Autoclave:

—on site No Desirable Yes Yes

—in laboratory room No No Desirable Yes

—double-ended No No Desirable Yes

Biological safety cabinets No Desirable Yes Yes

Personnel safety monitoring capabilitydNo No Desirable Yes

aEnvironmental and functional isolation from general traffic.

bDependent on location of exhaust (see Chapter 4).

cDependent on agent(s) used in the laboratory.

dFor example, window, closed-circuit television, two-way communication.

Thus, the assignment of a biosafety level takes into consideration the organism

(pathogenic agent) used, the facilities available, and the equipment practices and

procedures required to conduct work safely in the laboratory.

1. GENERAL PRINCIPLES

PART I

Biosafety guidelines

2. Microbiological

risk assessment

• 7 •

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 institution’s safety committee and biosafety personnel to ensure that appropriate

equipment and facilities are available to support the work being considered. Once

performed, risk assessments should be reviewed routinely and revised when necessary,

taking into consideration the 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 assess-

ment 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:

1. Pathogenicity of the agent and infectious dose

2. Potential outcome of exposure

3. Natural route of infection

4. Other routes of infection, resulting from laboratory manipulations (parenteral,

airborne, ingestion)

5. Stability of the agent in the environment

6. Concentration of the agent and volume of concentrated material to be manipulated

7. Presence of a suitable host (human or animal)

8. Information available from animal studies and reports of laboratory-acquired

infections or clinical reports

9. Laboratory activity planned (sonication, aerosolization, centrifugation, etc.)

10. Any genetic manipulation of the organism that may extend the host range of the

agent or alter the agent’s sensitivity to known, effective treatment regimens (see

Chapter 16)

11. Local availability of effective prophylaxis or therapeutic interventions.

• 8 •

LABORATORY BIOSAFETY MANUAL

On the basis of the information ascertained during the risk assessment, a biosafety

level can be assigned to the planned work, appropriate personal protective equipment

selected, and standard operating procedures (SOPs) incorporating other safety

interventions developed to ensure the safest possible conduct of the work.

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 cautious approach to specimen manipulation.

1. Standard precautions (2) should always be followed, and barrier protections applied

(gloves, gowns, eye protection), whenever samples are obtained from patients.

2. Basic containment – Biosafety Level 2 practices and procedures should be the

minimum requirement for handling specimens.

3. 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:

1. Medical data on the patient

2. Epidemiological data (morbidity and mortality data, suspected route of trans-

mission, other outbreak investigation data)

3. Information on the geographical origin of the specimen.

In the case of outbreaks of disease of unknown etiology, appropriate ad hoc guidelines

may be generated and posted by national competent authorities and/or WHO on the

Wor ld Wide Web (as was the case during the 2003 emergence of the severe acute

respiratory syndrome (SARS)) to indicate how specimens should be consigned for

shipment and the biosafety level at which they should be analysed.

Risk assessment and genetically modified microorganisms

A detailed discussion of risk assessment and genetically modified organisms (GMOs)

is provided in Chapter 16.

• 9 •

3. Basic laboratories –

Biosafety Levels 1 and 2

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 1–4. 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 (GMT).

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 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, standard precautions (2) 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 laboratories of all biosafety

levels. 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

This code is a listing of the most essential laboratory practices and procedures that are

basic to GMT. 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. GMT are fundamental to laboratory safety. Specialized laboratory

equipment is a supplement to but can never replace appropriate procedures. The most

important concepts are listed below.

Access

1. The international biohazard warning symbol and sign (Figure 1) must be displayed

on the doors of the rooms where microorganisms of Risk Group 2 or higher risk

groups are handled.

• 10 •

LABORATORY BIOSAFETY MANUAL

2. Only authorized persons should be allowed to enter the laboratory working areas.

3. Laboratory doors should be kept closed.

4. Children should not be authorized or allowed to enter laboratory working areas.

5. Access to animal houses should be specially authorized.

6. No animals should be admitted other than those involved in the work of 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, body fluids and other potentially 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.

Figure 1. Biohazard warning sign for laboratory doors

BIOHAZARD

ADMITTANCE TO AUTHORIZED PERSONNEL ONLY

Biosafety Level: _________________________________

Responsible Investigator: _________________________

In case of emergency call: ________________________

Daytime phone: __________Home phone: ___________

Authorization for entrance must be obtained from

the Responsible Investigator named above.

WHO 04.64

• 11 •

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 the laboratory, e.g.

in canteens, coffee rooms, offices, libraries, staff rooms and toilets.

6. Open-toed footwear must not be worn in laboratories.

7. Eating, drinking, smoking, 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 that has been used in the laboratory must not be

stored in the same lockers or cupboards as street clothing.

Procedures

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.

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.

7. Contaminated liquids must be decontaminated (chemically or physically) before

discharge to the sanitary sewer. An effluent treatment system may be required,

depending on the risk assessment for the agent(s) being handled.

8. Written documents that are expected to be removed from the laboratory need to

be protected from contamination while in the laboratory.

Laboratory working areas

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.

3. BASIC LABORATORIES – BIOSAFETY LEVELS 1 AND 2

• 12 •

LABORATORY BIOSAFETY MANUAL

Biosafety management

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. 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.

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:

1. Formation of aerosols

2. Work with large volumes and/or high concentrations of microorganisms

3. Overcrowding and too much equipment

4. Infestation with rodents and arthropods

5. Unauthorized entrance

6. Workflow: use of specific samples and reagents.

Examples of laboratory designs for Biosafety Levels 1 and 2 are shown in Figures 2

and 3, respectively.

Design features

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.

3. Bench tops should be 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.

• 13 •

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-washing basins, with running water if possible, should be provided in each

laboratory room, preferably near the exit door.

11. Doors should have vision panels, appropriate fire ratings, and preferably be self-

closing.

12. At Biosafety Level 2, an autoclave or other means of decontamination should be

available in appropriate proximity to 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

(see Annex 1).

3. BASIC LABORATORIES – BIOSAFETY LEVELS 1 AND 2

Figure 2. A typical Biosafety Level 1 laboratory

(graphics kindly provided by CUH2A, Princeton, NJ, USA)

• 14 •

LABORATORY BIOSAFETY MANUAL

15. 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 be fitted to 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. 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 (see Chapter 9).

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:

1. Designed to prevent or limit contact between the operator and the infectious

material

2. Constructed of materials that are impermeable to liquids, resistant to corrosion

and meet structural requirements

3. Fabricated to be free of burrs, sharp edges and unguarded moving parts

4. 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.

Detailed performance and construction specifications may need to be consulted to

ensure that the equipment possesses the necessary safety features (see also Chapters

10 and 11).

• 15 •

3. BASIC LABORATORIES – BIOSAFETY LEVELS 1 AND 2

Figure 3. A typical Biosafety Level 2 laboratory

(graphics kindly provided by CUH2A, Princeton, NJ, USA). Procedures likely to generate

aerosols are performed within a biological safety cabinet. Doors are kept closed and

are posted with appropriate hazard signs. Potentially contaminated wastes are separated

from the general waste stream.

Essential biosafety equipment

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 to reduce aerosol production.

• 16 •

LABORATORY BIOSAFETY MANUAL

4. Screw-capped tubes and bottles.

5. Autoclaves or other appropriate means 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 before being taken into use. Recertification should take place

at regular intervals, according to the manufacturer’s instructions (see Chapter 7).

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:

1. Provision of active or passive immunization where indicated (see Annex 2)

2. Facilitation of the early detection of laboratory-acquired infections

3. Exclusion of highly susceptible individuals (e.g. pregnant women or immuno-

compromised individuals) from highly hazardous laboratory work

4. Provision of effective personal protective equipment and procedures.

Guidelines for the surveillance of laboratory workers handling microorganisms

at Biosafety Level 1

Historical evidence indicates that the microorganisms handled at this level are unlikely

to cause human disease or animal disease of veterinary importance. Ideally, however,

all laboratory workers should undergo a pre-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

GMT.

Guidelines for the surveillance of laboratory workers handling microorganisms

at Biosafety Level 2

1. A pre-employment or preplacement health check is necessary. The person’s medical

history should be recorded and a targeted occupational health assessment

performed.

2. Records of illness and absence should be kept by the laboratory management.

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.

Training

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-

• 17 •

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, including the safety or

operations manual. 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 (see also Chapter 21).

Staff training should always include information on safe methods for highly

hazardous procedures that are commonly encountered by all laboratory personnel

and which involve:

1. Inhalation risks (i.e. aerosol production) when using loops, streaking agar plates,

pipetting, making smears, opening cultures, taking blood/serum samples,

centrifuging, etc.

2. Ingestion risks when handling specimens, smears and cultures

3. Risks of percutaneous exposures when using syringes and needles

4. Bites and scratches when handling animals

5. Handling of blood and other potentially hazardous pathological materials

6. 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:

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?

3. BASIC LABORATORIES – BIOSAFETY LEVELS 1 AND 2

• 18 •

LABORATORY BIOSAFETY MANUAL

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).

Handling and disposal procedures for contaminated materials and wastes

An identification and separation system for infectious materials and their containers

should be adopted. National and international regulations must be followed. Categories

should include:

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 disposal

container. Disposable syringes, used alone or with needles, should be placed in sharps

disposal containers and incinerated, with prior autoclaving if required.

Sharps disposal containers must be puncture-proof/-resistant and must 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 disposal containers must not be discarded in landfills.

Contaminated (potentially infectious) materials for autoclaving and reuse

No precleaning should be attempted of any contaminated (potentially infectious)

materials to be autoclaved and reused. Any necessary cleaning or repair must be done

only after autoclaving or disinfection.

Contaminated (potentially infectious) materials for disposal

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

• 19 •

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 incinerator. Reusable transfer containers should be

leakproof and have tight-fitting covers. They should be disinfected and cleaned before

they are returned to the laboratory for further use.

Discard containers, pans or jars, preferably unbreakable (e.g. plastic), should be

placed at every work station. When disinfectants are used, 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 discard

containers should be decontaminated and washed before reuse.

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 (see

section on Incineration in Chapter 14).

Chemical, fire, electrical, radiation and equipment safety

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, electrical and radiation hazards are considered in greater detail in Part VI of this

manual (Chapters 17 and 18).

Additional information regarding safety equipment is presented in Chapter 11.

3. BASIC LABORATORIES – BIOSAFETY LEVELS 1 AND 2

4. The containment laboratory –

Biosafety Level 3

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 pro-

grammes over and 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:

1. Code of practice

2. Laboratory design and facilities

3. Health and medical surveillance.

Laboratories in this category should be registered or listed with the national or other

appropriate health authorities.

Code of practice

The code of practice for basic laboratories – Biosafety Levels 1 and 2 applies except

where modified as follows.

1. The international biohazard warning symbol and sign (see Figure 1) displayed on

laboratory access doors must identify the biosafety level and the name of the

laboratory supervisor who controls access, and indicate any special conditions for

entry into the area, e.g. immunization.

2. 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, as are

sleeves that do not fully cover the forearms. Laboratory protective clothing must

not be worn outside the laboratory, and it must be decontaminated before it is

laundered. The removal of street clothing and change into dedicated laboratory

clothing may be warranted when working with certain agents (e.g. agricultural or

zoonotic agents).

• 20 •

3. Open manipulations of all potentially infectious material must be conducted within

a biological safety cabinet or other primary containment device (see also Chapter 10).

4. Respiratory protective equipment may be necessary for some laboratory procedures

or working with animals infected with certain pathogens (see Chapter 11).

Laboratory design and facilities

The laboratory design and facilities for basic laboratories – Biosafety Levels 1 and 2

apply except where modified as follows:

1. The laboratory must 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 (e.g. a double-door entry or basic laboratory –

Biosafety Level 2), describing a specific area designed to maintain the pressure

differential between the laboratory and its adjacent space. The anteroom should

have facilities for separating clean and dirty clothing and a shower may also be

necessary.

2. Anteroom doors may be self-closing and interlocking so that only one door is

open at a time. A break-through panel may be provided for emergency exit use.

3. Surfaces of walls, floors and ceilings should be water-resistant and easy to clean.

Openings through these surfaces (e.g. for service pipes) should be sealed to facilitate

decontamination of the room(s).

4. The laboratory room must be sealable for decontamination. Air-ducting systems

must be constructed to permit gaseous decontamination.

5. Windows must be closed, sealed and break-resistant.

6. A hand-washing station with hands-free controls should be provided near each

exit door.

7. There must be a controlled ventilation system that maintains a directional airflow

into the laboratory room. A visual monitoring device with or without alarm(s)

should be installed so that staff can at all times ensure that proper directional

airflow into the laboratory room is maintained.

8. The building ventilation system must be so constructed that air from the contain-

ment laboratory – Biosafety Level 3 is not recirculated to other areas within the

building. Air may be high-efficiency particulate air (HEPA) filtered, reconditioned

and recirculated within that laboratory. When exhaust air from the laboratory (other

than from biological safety cabinets) is discharged to the outside of the building, it

must be dispersed away from occupied buildings and air intakes. Depending on

the agents in use, this air may be discharged through HEPA filters. A heating,

ventilation and air-conditioning (HVAC) control system may be installed to prevent

sustained positive pressurization of the laboratory. Consideration should be given

to the installation of audible or clearly visible alarms to notify personnel of HVAC

system failure.

4. THE CONTAINMENT LABORATORY – BIOSAFETY LEVEL 3

• 21 •

• 22 •

LABORATORY BIOSAFETY MANUAL

9. All HEPA filters must be installed in a manner that permits gaseous

decontamination and testing.

10. Biological safety cabinets should be sited away from walking areas and out of cross-

currents from doors and ventilation systems (see Chapter 10).

11. The exhaust air from Class I or Class II biological safety cabinets (see Chapter 10),

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.

12. An autoclave for the decontamination of contaminated waste material should be

available in the containment laboratory. If infectious waste has to be removed from

the containment laboratory for decontamination and disposal, it must be

transported in sealed, unbreakable and leakproof containers according to national

or international regulations, as appropriate.

13. Backflow-precaution devices must be fitted to the water supply. Vacuum lines should

be protected with liquid disinfectant traps and HEPA filters, or their equivalent.

Alternative vacuum pumps should also be properly protected with traps and filters.

14. The containment laboratory – Biosafety Level 3 facility design and operational

procedures should be documented.

An example of laboratory design for Biosafety Level 3 is shown in Figure 4.

Laboratory equipment

The principles for the selection of laboratory equipment, including biological safety

cabinets (see Chapter 10) are the same as for the basic laboratory – Biosafety Level 2.

However, at Biosafety Level 3, manipulation of all potentially infectious material must

be conducted within a biological safety cabinet or other primary containment device.

Consideration should be given to equipment such as centrifuges, which will need

additional containment accessories, for example, safety buckets or containment rotors.

Some centrifuges and other equipment, such as cell-sorting instruments for use with

infected cells, may need additional local exhaust ventilation with HEPA filtration for

efficient containment.

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 containment

laboratories – Biosafety Level 3 is mandatory. This should include recording of a

detailed medical history and an occupationally-targeted physical examination.

2. After a satisfactory clinical assessment, the examinee may be provided with a medical

contact card (e.g. as shown in Figure 5) stating that he or she is employed in a

facility with a containment laboratory – Biosafety Level 3. This card should include

• 23 •

4. THE CONTAINMENT LABORATORY – BIOSAFETY LEVEL 3

Figure 4. A typical Biosafety Level 3 laboratory

(graphics kindly provided by CUH2A, Princeton, NJ, USA). The laboratory is separated

from general traffic flow and accessed through an anteroom (double door entry or

basic laboratory – Biosafety Level 2) or an airlock. An autoclave is available within the

facility for decontamination of wastes prior to disposal. A sink with hands-free operation

is available. Inward directional airflow is established and all work with infectious

materials is conducted within a biological safety cabinet.

a picture of the card holder, be wallet-sized, and always be carried by the holder.

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.

• 24 •

LABORATORY BIOSAFETY MANUAL

A. Front of card

ILLNESS SURVEILLANCE NOTICE

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.

Dr Tel (Work):

Tel (Home):

Dr Tel (Work):

Tel (Home):

B. Back of card

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.

Name of laboratory:

Address:

Tel:

Figure 5. Suggested format for medical contact card

Card holder’s

picture

• 25 •

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. The following information is intended only as

introductory material. Entities working to pursue development of a Biosafety Level 4

laboratory should contact the WHO Biosafety programme for additional information.1

Code of practice

The code of practice for Biosafety Level 3 applies except where modified as follows:

1. The two-person rule should apply, whereby no individual ever works alone. This is

particularly important if working in a Biosafety Level 4 suit facility.

2. A complete change of clothing and shoes is required prior to entering and upon

exiting the laboratory.

3. Personnel must be trained in emergency extraction procedures in the event of

personnel injury or illness.

4. A method of communication for routine and emergency contacts must be

established between personnel working within the maximum containment

laboratory – Biosafety Level 4 and support personnel outside the laboratory.

Laboratory design and facilities

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.

1. Primary containment. An efficient primary containment system must be in place,

consisting of one or a combination of the following.

—Class III cabinet laboratory. Passage through a minimum of two doors prior to

entering the rooms containing the Class III biological safety cabinet(s) (cabinet

room) is required. In this laboratory configuration the Class III biological safety

1Biosafety programme, Department of Communicable Disease Surveillance and Response, World Health

Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (http://www.who.int/csr/).

• 26 •

LABORATORY BIOSAFETY MANUAL

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 10).

—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 so as to direct personnel through

the changing and decontamination areas prior to entering areas where infectious

materials are manipulated. A suit decontamination shower must be provided

and used by 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, HEPA-filtered, 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 10).

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 pressure to

the surrounding laboratory at all times. A dedicated non-recirculating

ventilating system for the cabinet laboratory is required.

—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 airflow within the suit area from the area of least hazard to

• 27 •

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. Airflow 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 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.

7. Emergency power and dedicated power supply line(s) must be provided.

8. Containment drain(s) must be installed.

Because of the great complexity of the engineering, design and construction of Biosafety

Level 4 facilities, in either cabinet or suit configuration, schematic representations of

such facilities have not been included.

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

designated receiving hospitals, should also be involved.

5. THE MAXIMUM CONTAINMENT LABORATORY – BIOSAFETY LEVEL 4

6. Laboratory animal facilities

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

RISK GROUP CONTAINMENT LEVEL LABORATORY PRACTICES AND SAFETY EQUIPMENT

1ABSL-1 Limited access, protective clothing and gloves.

2ABSL-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.

3ABSL-3 ABSL-2 practices plus: controlled access. BSCs and

special protective clothing for all activities.

4ABSL-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.

ABSL, animal facility Biosafety Level; BSCs, biological safety cabinets

Animal facilities, like laboratories, may be designated according to a risk assessment

and the risk group of the microorganisms under investigation, as Animal facility

Biosafety Level 1, 2, 3 or 4.

With respect to agents to be used in the animal laboratory, factors for consideration

include:

1. The normal route of transmission

• 28 •

2. The volumes and concentrations to be used

3. The route of inoculation

4. Whether and by what route these agents may be excreted.

With respect to animals to be used in the animal laboratory, factors for consideration

include:

1. The nature of the animals, i.e. their aggressiveness and tendency to bite and scratch

2. Their natural ecto- and endoparasites

3. The zoonotic diseases to which they are susceptible

4. The possible dissemination of allergens.

As with laboratories, the requirements for design features, equipment and precautions

increase in stringency according to the animal 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. GMT are

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.

Animal facility – Biosafety Level 2

This is suitable for work with animals that are deliberately inoculated with micro-

organisms in Risk Group 2. The following safety precautions apply:

1. All the requirements for animal facilities – Biosafety Level 1 must be met.

2. Biohazard warning signs (see Figure 1) should be posted on doors and other

appropriate places.

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 airflow 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).

6. LABORATORY ANIMAL FACILITIES

• 29 •

• 30 •

LABORATORY BIOSAFETY MANUAL

12. Biological safety cabinets (Classes I or II) or isolator cages with dedicated air supplies

and HEPA-filtered exhaust air must be provided for work that may involve the

generation of aerosols.

13. An autoclave must be available on site or in appropriate proximity to the animal

facility.

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/-resistant 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 should be incinerated.

20. Protective clothing and equipment must be worn in the facility, and removed on

leaving.

21. Hand-washing facilities must be provided. Staff must wash their hands before

leaving the animal facility.

22. All injuries, however minor, must be treated appropriately, reported and recorded.

23. Eating, drinking, smoking and application of cosmetics must be forbidden in the

facility.

24. All personnel must receive appropriate training.

Animal facility – Biosafety Level 3

This is suitable for work with animals that are deliberately inoculated with agents in

Risk Group 3, or when otherwise indicated by a risk assessment. All systems, practices

and procedures need to be reviewed and recertified annually. The following safety

precautions apply:

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 a double-door entrance forming an anteroom.

4. Hand-washing facilities must be provided in the anteroom.

5. Showers should be provided in the anteroom.

6. There must be mechanical ventilation to ensure a continuous airflow 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.

7. 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.

• 31 •

8. An incinerator should be readily available on site or alternative arrangements should

be made with the authorities concerned.

9. Animals infected with Risk Group 3 microorganisms must be housed in cages in

isolators or rooms with ventilation exhausts placed behind the cages.

10. Bedding should be as dust-free as possible.

11. All protective clothing must be decontaminated before it is laundered.

12. Windows must be closed and sealed, and resistant to breakage.

13. Immunization of staff, as appropriate, should be offered.

Animal facility – Biosafety Level 4

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 over and above those described here (see

Chapter 5).

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 with 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

laboratories – Biosafety Level 4.

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. 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.

8. The facility must be ventilated by a HEPA-filtered exhaust system designed to ensure

a negative pressure (inward directional airflow).

9. The ventilation system must be designed to prevent reverse flow and positive-

pressurization.

10. A double-ended autoclave with the clean end in a room outside the containment

rooms must be provided for exchange of materials.

11. A pass-through airlock with the clean end in a room outside the containment rooms

must be provided for exchange of non-autoclavable 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 animal bedding and waste must be autoclaved before removal from the facility.

6. LABORATORY ANIMAL FACILITIES

• 32 •

LABORATORY BIOSAFETY MANUAL

15. There must be medical supervision of staff.

Invertebrates

As with vertebrates, the animal facility biosafety level will be determined by the risk

groups of the agents under investigation or when otherwise indicated by a risk

assessment. The following additional precautions are necessary with certain arthropods,

particularly with flying insects:

1. Separate rooms should be provided for infected and noninfected invertebrates.

2. The rooms should be capable of being sealed for fumigation.

3. Insecticide sprays should be readily available.

4. “Chilling” facilities should be provided to reduce, where necessary, the activity of

invertebrates.

5. Access should be through an anteroom containing insect traps and with arthropod-

proof screens on the doors.

6. All exhaust ventilation ducts and openable windows should be fitted with

arthropod-proof screens.

7. Waste traps on sinks and sluices should not be allowed to dry out.

8. All waste should be decontaminated by autoclaving, as some invertebrates are not

killed by all disinfectants.

9. A check should be kept on the numbers of larval and adult forms of flying, crawling

and jumping arthropods.

10. Containers for ticks and mites should stand in trays of oil.

11. Infected or potentially infected flying insects must be contained in double-netted

cages.

12. Infected or potentially infected arthropods must be handled in biological safety

cabinets or isolators.

13. Infected or potentially infected arthropods may be manipulated on cooling trays.

For further information see references (3–6).

• 33 •

7. Guidelines for

laboratory/facility

commissioning

Laboratory/facility commissioning may be defined as the systematic review and

documentation process signifying that specified laboratory structural components,

systems and/or system components have been installed, inspected, functionally tested

and verified to meet national or international standards, as appropriate. The respective

building system’s design criteria and design function establish these requirements. In

other words, laboratories designated as Biosafety Levels 1–4 will have different and

increasingly complex commissioning requirements. Geographical and climatic

conditions, such as geological fault lines or extreme heat, cold or humidity may also

affect the laboratory design and therefore the commissioning requirements. Upon the

completion of the commissioning process, the pertinent structural components and

support systems will have been subjected to the various operating conditions and failure

modes that can be reasonably expected, and will have been approved.

The commissioning process and acceptance criteria should be established early,

preferably during the programming phase of the construction or renovation project.

By acknowledging the commissioning process early in the project, architects, engineers,

safety and health personnel and ultimately the laboratory occupants understand the

performance requirements of the specific laboratory and set uniform expectations for

laboratory and/or facility performance. The commissioning process provides the

institution and the surrounding community with a greater degree of confidence that

the structural, electrical, mechanical and plumbing systems, containment and

decontamination systems, and security and alarm systems will operate as designed, to

assure containment of any potentially dangerous microorganisms being worked with

in a particular laboratory or animal facility.

Commissioning activities generally begin during the programming phase of the

project and proceed through the construction and subsequent warranty period for

the laboratory/facility. Warranty periods should generally extend for one year following

occupancy. It is recommended that a commissioning agent is retained who is

independent of the architectural, engineering and construction firms involved in the

design and construction. The commissioning agent serves as an advocate for the

institution constructing or renovating the laboratory and should be considered as a

member of the design team; involvement of the agent in the early programming phase

of the project is essential. In some cases, the institution may act as its own

commissioning agent. In the case of more complex laboratory facilities (Biosafety

• 34 •

LABORATORY BIOSAFETY MANUAL

Levels 3 or 4), the institution may wish to retain an outside commissioning agent who

has demonstrated experience and success in the commissioning of complex biosafety

laboratory and animal facilities. When an independent commissioning agent is used,

the institution should still be a member of the commissioning team. It is recommended

that, in addition to the commissioning agent, the institution’s Safety Officer, Project

Officer, Programme Manager and a representative of the Operations and Maintenance

staff are also part of the team.

The following is a list of laboratory systems and components that may be included

in a commissioning plan for functional testing, depending on the containment level

of the facility being renovated or constructed. The list is not exhaustive. Obviously,

the actual commissioning plan will reflect the complexity of the laboratory being

planned.

1. Building automation systems including links to remote monitoring and control

sites

2. Electronic surveillance and detection systems

3. Electronic security locks and proximity device readers

4. Heating, ventilation (supply and exhaust) and air-conditioning (HVAC) systems

5. High-efficiency particulate air (HEPA) filtration systems

6. HEPA decontamination systems

7. HVAC and exhaust air system controls and control interlocks

8. Airtight isolation dampers

9. Laboratory refrigeration systems

10. Boilers and steam systems

11. Fire detection, suppression and alarm systems

12. Domestic water backflow prevention devices

13. Processed water systems (i.e. reverse osmosis, distilled water)

14. Liquid effluent treatment and neutralization systems

15. Plumbing drain primer systems

16. Chemical decontaminant systems

17. Medical laboratory gas systems

18. Breathing air systems

19. Service and instrument air systems

20. Cascading pressure differential verification of laboratories and support areas

21. Local area network (LAN) and computer data systems

22. Normal power systems

23. Emergency power systems

24. Uninterruptible power systems

25. Emergency lighting systems

26. Lighting fixture penetration seals

27. Electrical and mechanical penetration seals

28. Telephone systems

• 35 •

29. Airlock door control interlocks

30. Airtight door seals

31. Window and vision-panel penetration seals

32. Barrier pass-through penetration

33. Structural integrity verification: concrete floors, walls and ceilings

34. Barrier coating verification: floors, walls and ceilings

35. Biosafety Level 4 containment envelope pressurization and isolation functions

36. Biological safety cabinets

37. Autoclaves

38. Liquid nitrogen system and alarms

39. Water detection systems (e.g. in case of flooding inside containment zone)

40. Decontamination shower and chemical additive systems

41. Cage-wash and neutralization systems

42. Waste management.

7. GUIDELINES FOR LABORATORY/FACILITY COMMISSIONING

8. Guidelines for

laboratory/facility

certification

Laboratories are complex and dynamic environments. Today’s biomedical research

and clinical laboratories must be able to adapt quickly to continuously increasing public

health needs and pressures. An example of this is the need for laboratories to adjust

priorities to meet the challenges of emerging or re-emerging infectious diseases. In

order to assure that adaptation and maintenance are undertaken promptly and in an

appropriate and safe manner, all biological research and clinical laboratories should

be regularly certified. Laboratory certification helps to ensure that:

1. Proper engineering controls are being used and are functioning adequately as

designed

2. Appropriate site and protocol specific administrative controls are in place

3. Personal protective equipment is appropriate for the tasks being performed

4. Decontamination of waste and materials has been adequately considered and proper

waste management procedures are in place

5. Proper procedures for general laboratory safety, including physical, electrical and

chemical safety are in place.

Laboratory certification differs from laboratory commissioning activities (Chapter 7)

in several important ways. Laboratory certification is the systematic examination of

all safety features and processes within the laboratory (engineering controls, personal

protective equipment and administrative controls). Biosafety practices and procedures

are also examined. Laboratory certification is an on-going quality and safety assurance

activity that should take place on a regular basis.

Adequately trained safety and health or biosafety professionals may conduct

laboratory certification activities. Institutions may employ personnel having the

appropriate skill-set required for conducting audits, surveys or inspections (these terms

are used interchangeably) associated with the certification process. However,

institutions may consider engaging or be required to engage a third party to provide

these services.

Biomedical research and clinical laboratory facilities may develop audit, survey or

inspection tools to help ensure consistency in the certification process. These tools

should be flexible enough to allow for the physical and procedural differences between

laboratories necessitated by the type of work being conducted, while at the same time

providing a consistent approach within the institution. Care must be taken to ensure

• 36 •

that these tools are used only by appropriately trained personnel, and that they are not

used as a substitute for a sound professional biosafety assessment. Examples of such

tools are provided in Tables 5–7.

Findings of the audit, survey or inspection should be discussed with laboratory

personnel and management. Within the laboratory, an individual should be identified

and made responsible for ensuring that corrective actions are taken for all deficiencies

identified during the audit process. Certification of the laboratory should not be

completed, and the laboratory should not be declared functional, until deficiencies

have been adequately addressed.

The complexity of Biosafety Level 4 laboratory operations goes beyond the scope

of this manual. For details and further information, please contact the WHO Biosafety

programme1 (see also Annex 3).

8. GUIDELINES FOR LABORATORY/FACILITY CERTIFICATION

1WHO Biosafety programme, Department of Communicable Disease Surveillance and Response, World

Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (http://www.who.int/csr/).

• 37 •

• 38 •

LABORATORY BIOSAFETY MANUAL

Table 5. Basic Laboratory – Biosafety Level 1: laboratory safety survey

Location Date

Person in charge of laboratory

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

Laboratory

Proper signage: ultraviolet light, laser,

radioactive material, etc. ........................ 

Appropriate biosafety guidelines available

and followed ........................................... 

Laboratory equipment properly labelled

(biohazardous, radioactive, toxic, etc.) .. 

Laboratory design

Designed for easy cleaning ......................... 

Room ultraviolet lights on interlock switch 

All shelves secured ..................................... 

Bench-tops waterproof and resistant to

acids, alkali, organic solvents and heat . 

Adequate illumination provided .................. 

Adequate storage space available and

appropriately used ................................. 

Gas cylinders

All cylinders secured ................................... 

Caps on reserve cylinders ........................... 

Asphyxiating and hazardous gases only in

ventilated rooms .................................... 

Excess or empty cylinders present ............. 

Chemicals

Flammables stored in flammable storage

cabinet .................................................... 

Peroxide formers double-dated (received

and opened) ........................................... 

Chemicals properly segregated................... 

Hazardous chemicals stored above eye

level ........................................................ 

Chemicals stored on the floor ..................... 

Chemical containers left open ..................... 

All solutions properly labelled..................... 

Mercury thermometers in use .................... 

Refrigerators/freezers/cold rooms

Food for human consumption present ....... 

Flammables in explosion-proof/-safe units 

Labelled externally if containing

carcinogens, radioactivity and/or

biohazards .............................................. 

Cold-room has emergency release ............. 

Biosafety Level:

Attach the appropriate

Biosafety Level Survey

Form

• 39 •

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

Electrical equipment

Extension cords present ............................. 

Outlets earthed/grounded and with

proper polarity ....................................... 

Connections by sinks, under showers,

etc. ......................................................... 

Equipment with frayed or damaged

wiring ..................................................... 

Overloaded outlets or electrical strips ........ 

Power strips mounted off the floor ............. 

Proper fuses in conduits ............................. 

Electrical outlets near water sources meet

local codes ............................................. 

Earths/grounds present on electrical cords 

Portable space heaters ................................ 

Personal protective equipment

Eyewash available in laboratory .................. 

Safety shower available .............................. 

Personal protective equipment available

(gloves, gowns, goggles, etc.) ............... 

Occupants properly attired ......................... 

Laboratory coats, gowns, smocks, gloves

and other personal protective clothing

not worn outside the laboratory ............. 

Personal protective equipment available

for cryogenic storage ............................. 

Waste management

Evidence of improper waste disposal ......... 

Wastes segregated in proper containers .... 

Chemical waste containers tagged, labelled,

dated and kept closed ............................ 

Chemical waste containers appropriately

handled and stored ................................ 

Sharps containers used and disposed of

properly .................................................. 

No trash on floor ......................................... 

Waste disposal procedures posted in

laboratory ............................................... 

Occupational health and safety programmes available

Hazard communication ............................... 

Respiratory protection ................................ 

Hearing conservation .................................. 

Formaldehyde monitoring ........................... 

Ethylene oxide monitoring .......................... 

Anaesthetic gas monitoring ........................ 

8. GUIDELINES FOR LABORATORY/FACILITY CERTIFICATION

• 40 •

LABORATORY BIOSAFETY MANUAL

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

General engineering controls

Laboratory airflow is negative to general

occupancy, corridor and office areas ..... 

Cup sinks or drains acting as vents ............ 

Sink available for hand-washing ................. 

Exposed machine parts (pulleys, gears) ..... 

Vacuum line has filters and traps on

laboratory benches ................................ 

Backflow hazards to water supply .............. 

Distilled water systems in good condition .. 

Active and effective arthropod and rodent

control programme ................................ 

General practices and procedures

Food for human consumption stored

outside the laboratory ............................ 

Microwave oven(s) clearly labelled “No

Food Preparation, Laboratory Use Only” 

Eating, drinking, smoking and/or applying

of cosmetics occurring in the laboratory 

Pressurized glass containers taped or

shielded (i.e. vacuum traps) .................. 

Mouth pipetting prohibited ......................... 

Mechanical pipetting devices available

and used ................................................. 

Protective laboratory clothing stored

separately from street clothing .............. 

General laboratory housekeeping

Glass containers stored on the floor ........... 

Trip hazards evident .................................... 

Clean absorbent pads on work surfaces ..... 

Broken glassware handled by mechanical

means (brush and dustpan, tongs, etc.) 

Fire protection

Sprinkler heads free and unobstructed ....... 

Open penetrations in walls, ceiling, floor, etc. 

Wiring or tubing through door openings .... 

Minimum passage width of 1 m in laboratory 

Storage observed on ductwork or light fixtures 

Excess combustibles stored in laboratory .. 

Heated constant temperature baths

Equipped with low water level and

overheat shutoff ..................................... 

Constructed of noncombustible materials .. 

Safety surveyor’s signature: Date survey completed:

• 41 •

Table 6. Basic laboratory – Biosafety Level 2: laboratory safety survey.

This form is used in conjunction with the Biosafety Level 1 laboratory safety

survey form

Location Date

Person in charge of laboratory

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

Biological safety cabinet (BSC) Date:

Certification within last year ....................... 

BSC surface wiped down with appropriate .Location:

disinfectant at beginning and end of

each procedure ......................................  Brand:

Front grill and exhaust filter unobstructed.. 

Open flames used inside cabinet ................  Type:

Vacuum lines have in-line filters and

disinfectant traps in use .........................  Serial no.:

BSC compromised by room air or location 

BSC used when there is potential for

creating aerosols .................................... 

Laboratory

Access limited and restricted to authorized

personnel ............................................... 

Entry limited to personnel advised of all

potential hazards .................................... 

Biohazard sign posted on laboratory door

as appropriate ........................................ 

•Information on sign accurate and

current .............................................. 

•Sign legible and not defaced ............. 

All doors closed .......................................... 

Decontamination

Decontaminant specific to the organism(s)

in use...................................................... 

All spills and accidents involving infectious

materials reported to the laboratory

supervisor .............................................. 

Appropriate decontaminant used during

spill clean-ups ........................................ 

Work surfaces decontaminated before and

after each procedure, daily and after

spills ....................................................... 

Handling of contaminated waste

Infectious waste containers properly used .... 

Containers not overfilled ............................. 

Containers properly labelled and closed ..... 

Culture stocks and other regulated waste

properly decontaminated before disposal 

8. GUIDELINES FOR LABORATORY/FACILITY CERTIFICATION

• 42 •

LABORATORY BIOSAFETY MANUAL

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

Materials decontaminated outside the

laboratory transported in closed,

durable, leakproof containers according

to local rules and regulations ................. 

Mixed waste biologically decontaminated

prior to disposal as chemical or

radiological waste .................................. 

Personal protection

Laboratory personnel reminded of

appropriate immunizations/tests for

agents handled ....................................... 

Appropriate medical services contacted for

medical evaluations, surveillance and

treatment of occupational exposures ..... 

Gloves worn when handling infectious

material or contaminated equipment ..... 

Face protection provided when working

outside the BSC with infectious material 

Hands washed after removing gloves, after

working with infectious agents, before

leaving the laboratory ............................ 

Antimicrobial agent available for immediate

first aid ................................................... 

Practices

BSC used when potential for creating

infectious aerosols/splashes exists ....... 

Biosafety manual prepared and adopted .... 

Personnel read, review and follow the

instructions on practices and procedures,

including safety or operations manual

(required for all personnel annually)...... 

Procedures performed so as to minimize

aerosols/splashes .................................. 

Needle-locking syringes/single-use needle-

syringe units used with infectious agents 

Centrifuge cups and rotors opened only in

a BSC...................................................... 

Infectious specimens transported outside

a BSC in approved containers following

approved transport regulations.............. 

Facility

Hand-washing sink available near

laboratory exit ........................................ 

Safety surveyor’s signature: Date survey completed:

• 43 •

Table 7. Containment laboratory – Biosafety Level 3: laboratory safety survey.

This form is used in conjunction with the Bioafety Level 1 and Biosafety

Level 2 laboratory safety survey forms

Location Date

Person in charge of laboratory

CHECKED ITEM (ENTER DATE OF CHECK) YES NO N/A COMMENTS

Facility

Laboratory separated from unrestricted

traffic flow in building ............................ 

Access to laboratory through an anteroom

with self-closing doors .......................... 

All penetrations in laboratory sealed or

sealable for decontamination ................. 

Room exhaust air single-pass and

exhausted away from occupied areas .... 

Controlled ventilation system to monitor

directional airflow available .................... 

Personal protection

Closed-front gowns worn in laboratory ...... 

Protective laboratory clothing worn only in

laboratory areas ..................................... 

Hand-washing sink foot, elbow or

automatically controlled ......................... 

Hand protection

Double gloves worn when handling infectious

material, potentially contaminated

equipment and work surfaces ................ 

Respiratory protection

Respiratory protection worn by all personnel

in the laboratory when aerosols are not

safely contained in a BSC ....................... 

Practices

Mucous membrane protection provided

when working with infectious material

outside a BSC ......................................... 

Personnel advised of special hazards

associated with the agent(s) .................. 

Personnel required to read and follow all

instructions on practices and procedures,

including safety or operations manual ... 

Personnel receive annual updates/additional

training for procedural changes ............. 

All contaminated waste autoclaved prior to

disposal .................................................. 

Safety surveyor’s signature: Date survey completed:

8. GUIDELINES FOR LABORATORY/FACILITY CERTIFICATION

PART II

Laboratory biosecurity

9. Laboratory biosecurity concepts

• 47 •

The Laboratory biosafety manual has in the past focused on traditional biosafety

guidance for laboratories. The manual emphasizes the use of good microbiological

work practices, appropriate containment equipment, proper facility design, operation

and maintenance, and administrative considerations to minimize the risk of worker

injury or illness. In following these recommendations, the risk to the environment

and surrounding community-at-large is also minimized. It has now become necessary

to expand this traditional approach to biosafety through the introduction of laboratory

biosecurity measures. Global events in the recent past have highlighted the need to

protect laboratories and the materials they contain from being intentionally

compromised in ways that may harm people, livestock, agriculture or the environment.

Before the laboratory biosecurity needs of a facility can be defined, however, it is

important to understand the distinction between “laboratory biosafety” and “laboratory

biosecurity”.

“Laboratory biosafety” is the term used to describe the containment principles,

technologies and practices that are implemented to prevent unintentional exposure

to pathogens and toxins, or their accidental release. “Laboratory biosecurity” refers to

institutional and personal security measures designed to prevent the loss, theft, misuse,

diversion or intentional release of pathogens and toxins.

Effective biosafety practices are the very foundation of laboratory biosecurity

activities. Through risk assessments, performed as an integral part of an institution’s

biosafety programme, information is gathered regarding the type of organisms

available, their physical location, the personnel who require access to them, and the

identification of those responsible for them. This information can be used to assess

whether an institution possesses biological materials that are attractive to those who

may wish to use them improperly. National standards should be developed that

recognize and address the ongoing responsibility of countries and institutions to protect

specimens, pathogens and toxins from misuse.

A specific laboratory biosecurity programme must be prepared and implemented

for each facility according to the requirements of the facility, the type of laboratory

work conducted, and the local conditions. Consequently, laboratory biosecurity

activities should be representative of the institution’s various needs and should include

input from scientific directors, principal investigators, biosafety officers, laboratory

• 48 •

LABORATORY BIOSAFETY MANUAL

scientific staff, maintenance staff, administrators, information technology staff, and

law enforcement agencies and security staff if appropriate.

Laboratory biosecurity measures should be based on a comprehensive programme

of accountability for pathogens and toxins that includes an updated inventory with

storage location, identification of personnel with access, description of use,

documentation of internal and external transfers within and between facilities, and

any inactivation and/or disposal of the materials. Likewise, an institutional laboratory

biosecurity protocol should be established for identifying, reporting, investigating and

remediating breaches in laboratory biosecurity, including discrepancies in inventory

results. The involvement and roles and responsibilities of public health and security

authorities in the event of a security infraction must be clearly defined.

Laboratory biosecurity training, distinct from laboratory biosafety training, should

be provided to all personnel. Such training should help personnel understand the

need for protection of such materials and the rationale for the specific biosecurity

measures, and should include a review of relevant national standards and institution-

specific procedures. Procedures describing the security roles and responsibilities of

personnel in the event of a security infraction should also be presented during training.

The professional and ethical suitability for working with dangerous pathogens of

all personnel who have regular authorized access to sensitive materials is also central

to effective laboratory biosecurity activities.

In summary, security precautions should become a routine part of laboratory work,

just as have aseptic techniques and other safe microbiological practices. Laboratory

biosecurity measures should not hinder the efficient sharing of reference materials,

clinical and epidemiological specimens and related information necessary for clinical

or public health investigations. Competent security management should not unduly

interfere with the day-to-day activities of scientific personnel or be an impediment to

conducting research. Legitimate access to important research and clinical materials

must be preserved. Assessment of the suitability of personnel, security-specific training

and rigorous adherence to pathogen protection procedures are reasonable means of

enhancing laboratory biosecurity. All such efforts must be established and maintained

through regular risk and threat assessments, and regular review and updating of

procedures. Checks for compliance with these procedures, with clear instructions on

roles, responsibilities and remedial actions, should be integral to laboratory biosecurity

programmes and national standards for laboratory biosecurity.

PART III

Laboratory equipment

10. Biological safety cabinets

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 onto a surface or into another liquid. Other

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 µm in diameter and small droplets of 5–100 µm

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. BSCs also protect the environment.

Over the years the basic design of BSCs has undergone several 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 µm 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 8.

Note. Horizontal and vertical outflow cabinets (“clean-air work stations”) are not

biological safety cabinets and should not be used as such.

Class I biological safety cabinet

Figure 6 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

• 51 •

• 52 •

LABORATORY BIOSAFETY MANUAL

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 operator’s

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

Table 8. Selection of a biological safety cabinet (BSC), by type of protection needed

TYPE OF PROTECTION BSC SELECTION

Personnel protection, microorganisms Class I, Class II, Class III

in Risk Groups 1–3

Personnel protection, microorganisms Class III

in Risk Group 4, glove-box laboratory

Personnel protection, microorganisms Class I, Class II

in Risk Group 4, suit laboratory

Product protection Class II, Class III only if laminar flow included

Volatile radionuclide/chemical protection, Class IIB1, Class IIA2 vented to the outside

minute amounts

Volatile radionuclide/chemical protection Class I, Class IIB2, Class III

Figure 6. Schematic diagram of a Class I biological safety cabinet.

A, front opening; B, sash; C, exhaust HEPA filter; D, exhaust plenum.

room air

potentially contaminated air

HEPA-filtered air

WHO 02.135

side view

• 53 •

10. BIOLOGICAL SAFETY CABINETS

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, 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.

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 air from a HEPA-filtered (sterile) supply 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 A1 BSC is shown in Figure 7. 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 flowing downwards

over the work surface. As the air flows downwards it “splits” about 6–18 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 airflow 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

• 54 •

LABORATORY BIOSAFETY MANUAL

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 8).

Class II type A2 vented to the outside, B1 and B2 biological safety cabinets

Class IIA2 vented to the outside, IIB1 (Figure 8) and IIB2 BSCs are variations of the

type IIA1. Their characteristics, along with those of Class I and Class III BSCs, are

indicated in Table 9. Each variation allows the BSC to be used for specialized purposes

(see Table 8). 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 (7) and (8), and from manufacturers’ brochures.

Figure 7. 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.

WHO 02.137

front view side view

room air

potentially contaminated air

HEPA-filtered air

• 55 •

10. BIOLOGICAL SAFETY CABINETS

Figure 8. 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 the

cabinet exhaust to the building exhaust air system is required.

Table 9. Differences between Class I, II and III biological safety cabinets (BSCs)

BSC FACE VELOCITY (m/s) AIRFLOW (%) EXHAUST SYSTEM

RECIRCULATED EXHAUSTED

Class Ia0.36 0 100 Hard duct

Class IIA1 0.38–0.51 70 30 Exhaust to room or thimble

connection

Class IIA2 0.51 70 30 Exhaust to room or thimble

vented to the connection

outsidea

Class IIB1a0.51 30 70 Hard duct

Class IIB2a0.51 0 100 Hard duct

Class IIIaNA 0 100 Hard duct

NA, not applicable.

aAll biologically contaminated ducts are under negative pressure or are surrounded by negative pressure

ducts and plenums.

WHO 02.138

front view side view

room air

potentially contaminated air

HEPA-filtered air

• 56 •

LABORATORY BIOSAFETY MANUAL

Class III biological safety cabinet

This type (Figure 9) 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. Airflow 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 can be sterilized and is equipped with a HEPA-filtered

exhaust. The Class III 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.

Biological safety cabinet air connections

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,

Figure 9. 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 the cabinet exhaust to an independent building exhaust air

system is required.

WHO 02.136

room air

potentially contaminated air

HEPA-filtered air

front view side view

CDC

• 57 •

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 airflow 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 be precisely matched to the airflow

requirements specified by the manufacturer for 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 1–4 microorganisms;

personnel protection against exposure to radionuclides and volatile toxic chemicals;

or a combination of these. Table 8 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 BSCs that are not ducted to building exhaust systems, or

Class IIA1 or Class IIA2 cabinets. Class IIB1 BSCs are acceptable for work with minute

amounts of volatile chemicals and radionuclides. A Class IIB2 BSC, 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 30–35 cm above the cabinet may

be required to provide for accurate air velocity measurement across the exhaust filter

and for exhaust filter changes.

Operators

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

10. BIOLOGICAL SAFETY CABINETS

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LABORATORY BIOSAFETY MANUAL

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.

Material placement

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 cabinet’s 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 airflow 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.

• 59 •

Open flames

Open flames should be avoided in the near microbe-free environment created inside

the BSC. They disrupt the airflow 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 laboratory’s 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, clean-up 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.

Certification

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 manufacturer’s 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.

Cleaning and disinfection

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 is left running. If not, it should be run for 5 min

in order to purge the atmosphere inside before it is switched off.

Decontamination

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.

10. BIOLOGICAL SAFETY CABINETS

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LABORATORY BIOSAFETY MANUAL

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 investigator’s 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. Airflow alarms indicate a disruption in the cabinet’s normal

airflow pattern. This represents an immediate danger to the operator or product. When

an airflow 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.

For further information see references (5) and (7–16), and Chapter 11.

• 61 •

11. Safety equipment

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 10.

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 10 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 12.

Information on equipment and operations that may create hazards is presented in

Annex 4.

Negative-pressure flexible-film isolators

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 to manipulate high-risk organisms (Risk Groups 3

or 4) in field work where it is not feasible or appropriate to install or maintain

conventional biological safety cabinets.

• 62 •

LABORATORY BIOSAFETY MANUAL

Table 10. Biosafety equipment

EQUIPMENT HAZARD CORRECTED SAFETY FEATURES

Biological safety

cabinet

— Class I Aerosol and spatter • Minimum inward airflow (face

velocity) at work access opening.

Adequate filtration of exhaust air.

•Does not provide product protection

— Class II Aerosol and spatter • Minimum inward airflow (face

velocity) at work access opening.

Adequate filtration of exhaust air

•Provides product protection

— Class III Aerosol and spatter • Maximum containment

•Provides product protection if

laminar flow air is included

Negative pressure Aerosol and spatter • Maximum containment

flexible-film isolator

Spatter shield Spatter of chemicals • Forms screen between operator and

work

Pipetting aids Hazards from pipetting by • Ease of use

mouth, e.g. ingestion of • Controls contamination of suction

pathogens, inhalation of end of pipette, protecting pipetting

aerosols produced by mouth aid, user and vacuum line

suction on pipette, blowing • Can be sterilized

out of liquid or dripping from • Controls leakage from pipette tip

pipette, contamination of

suction end of pipette

Loop microinciner- Spatter from transfer loops • Shielded in open-ended glass or

ators, disposable ceramic tube. Heated by gas or

loops electricity

•Disposable, no heating necessary

Leakproof vessels Aerosols, spillage and • Leakproof construction with lid or

for collection and leakage cover

transport of • Durable

infectious materials • Autoclavable

for sterilization

within a facility

Sharps disposal Puncture wounds • Autoclavable

containers • Robust, puncture-proof

Transport containers Release of microorganisms • Robust

between laboratories, • Watertight primary and secondary

institutions containers to contain spills

•Absorbent material to contain spills

• 63 •

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.

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. 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

Domestic (kitchen) homogenizers are not sealed and release aerosols. Only equipment

designed for laboratory use should be used. Their construction minimizes or prevents

11. SAFETY EQUIPMENT

EQUIPMENT HAZARD CORRECTED SAFETY FEATURES

Autoclaves, manual Infectious material (made • Approved design

or automatic safe for disposal or reuse) • Effective heat sterilization

Screw-capped bottles Aerosols and spillage • Effective containment

Vacuum line Contamination of laboratory • Cartridge-type filter prevents

protection vacuum system with passage of aerosols (particle size

aerosols and overflow fluids 0.45 µm)

•Overflow flask contains appropriate

disinfectant. Rubber bulb may be

used to close off vacuum auto

matically when storage flask is full

•Entire unit autoclavable

• 64 •

LABORATORY BIOSAFETY MANUAL

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.

Disposable transfer loops

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 airflow. These loops should be placed in

disinfectant after use and discarded as contaminated waste (see Chapter 3).

Microincinerators

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 can disturb the airflow and should therefore be

placed towards the back of the work surface in biological safety cabinets.

Personal protective equipment and clothing

Personal protective equipment and clothing 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 11

summarizes some personal protective equipment used in laboratories and the

protection afforded.

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 at the biological safety

cabinet. Aprons may 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. Laundering services should be provided at/near the facility.

Laboratory coats, gowns, coveralls, or aprons should not be worn outside the

laboratory areas.

Goggles, safety spectacles, face shields

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

• 65 •

11. SAFETY EQUIPMENT

Table 11. Personal protective equipment

EQUIPMENT HAZARD CORRECTED SAFETY FEATURES

Laboratory coats, Contamination of clothing • Back opening

gowns, coveralls • Cover street clothing

Plastic aprons Contamination of clothing • Waterproof

Footwear Impact and splash • Closed-toe

Goggles Impact and splash • Impact-resistant lenses (must be optically

correct or worn over corrective eye glasses)

•Side shields

Safety spectacles Impact • Impact-resistant lenses (must be optically

correct)

•Side shields

Face shields Impact and splash • Shield entire face

•Easily removable in case of accident

Respirators Inhalation of aerosols • Designs available include single-use

disposable; full-face or half-face air

purifying; full-face or hooded powered air

purifying (PAPR); and supplied air

respirators

Gloves Direct contact with • Disposable microbiologically approved latex,

microorganisms vinyl or nitrile

•Hand protection

Cuts • Mesh

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). Safety spectacles do not provide for adequate splash protection even when

side shields are worn with them. Goggles for splash and impact protection should be

worn over normal prescription eye glasses and contact lenses (which do not provide

protection against biological or chemical hazards). Face shields (visors) are made of

shatterproof plastic, fit over the face and are held in place by head straps or caps.

Goggles, safety spectacles, or face shields should not be worn outside the laboratory

areas.

Respirators

Respiratory protection may be used when carrying out high-hazard procedures (e.g.

cleaning up a spill of infectious material). The choice of respirator will depend on the

type of hazard(s). Respirators are available with interchangeable filters for protection

against gases, vapours, particulates and microorganisms. It is imperative that the filter

is fitted in the correct type of respirator. To achieve optimal protection, respirators

should be individually fitted to the operator’s 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. Surgical type masks are designed solely for patient protection and

do not provide respiratory protection to workers. Some single-use disposable

respirators (ISO 13.340.30) have been designed for protection against exposures to

biological agents.

Respirators should not be worn outside the laboratory areas.

Gloves

Contamination of hands may occur when laboratory procedures are performed. Hands

are also vulnerable to “sharps” injuries. Disposable microbiologically approved latex,

vinyl or nitrile 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 to powdered latex gloves should be available.

Stainless steel mesh gloves should be worn when there is a potential exposure to

sharp instruments e.g. during postmortem examinations. Such gloves protect against

slicing motion but do not protect against puncture injury.

Gloves should not be worn outside the laboratory areas.

For further information see references (12), (17) and (18).

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LABORATORY BIOSAFETY MANUAL

PART IV

Good microbiological

techniques

12. Laboratory techniques

• 69 •

Human error, poor laboratory techniques and misuse of equipment cause the majority

of laboratory 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

Improper collection, transport and handling of specimens in the laboratory carry a

risk of infection to the personnel involved.

Specimen containers

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.

Transport of specimens within the facility

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.

Receipt of specimens

Laboratories that receive large numbers of specimens should designate a particular

room or area for this purpose.

Opening packages

Personnel who receive and unpack specimens should be aware of the potential health

hazards involved, and should be trained to adopt standard precautions (2), particularly

when dealing with broken or leaking containers. Primary specimen containers should

be opened in a biological safety cabinet. Disinfectants should be available.

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LABORATORY BIOSAFETY MANUAL

Use of pipettes and pipetting aids

1. A pipetting aid must always be used. Pipetting by mouth must be prohibited.

2. All pipettes should have cotton plugs to reduce contamination of pipetting devices.

3. Air should never be blown through a liquid containing infectious agents.

4. Infectious materials should not be mixed by alternate suction and expulsion through

a pipette.

5. Liquids should not be forcibly expelled from pipettes.

6. Mark-to-mark pipettes are preferable to other types as they do not require expulsion

of the last drop.

7. Contaminated pipettes should be completely submerged in a suitable disinfectant

contained in an unbreakable container. They should be left in the disinfectant for

the appropriate length of time before disposal.

8. A discard container for pipettes should be placed within the biological safety cabinet,

not outside it.

9. Syringes fitted with hypodermic needles must not be used for pipetting.

10. Devices for opening septum-capped bottles that allow pipettes to be used and avoid

the use of hypodermic needles and syringes should be used.

11. To avoid dispersion of infectious material dropped from a pipette, an absorbent

material should be placed on the working surface; this should be disposed of as

infectious waste after use.

Avoiding the dispersal of infectious materials

1. In order to avoid the premature shedding of their loads, microbiological transfer

loops should have a diameter of 2–3 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. Care should be taken when drying sputum samples, to avoid creating aerosols.

4. Discarded specimens and cultures for autoclaving and/or disposal should be placed

in leakproof containers, e.g. laboratory discard bags. Tops should be secured (e.g.

with autoclave tape) prior to disposal into waste containers.

5. Working areas must be decontaminated with a suitable disinfectant at the end of

each work period.

For further information see reference (12).

Use of biological safety cabinets

1. The use and limitations of biological safety cabinets should be explained to all

potential users (see Chapter 10), 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.

• 71 •

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.

5. Bunsen burners must not be used in the cabinet. The heat produced will distort

the airflow 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 airflow by repeated removal and reintroduction

of his or her arms.

9. Air grills must not be blocked with notes, pipettes or other materials, as this will

disrupt the airflow 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.

12. Paperwork should never be placed inside biological safety cabinets.

For further information about biological safety cabinets see Chapter 10.

Avoiding ingestion of infectious materials and contact with skin and eyes

1. Large particles and droplets (> 5 µm 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. No articles should be placed in the mouth – pens, pencils, chewing gum – in the

laboratory.

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

1. Accidental inoculation resulting from injury with broken or chipped glassware

can be avoided through careful practices and procedures. Glassware should be

replaced with plastic ware whenever possible.

2. Accidental injection may result from sharps injuries e.g. with hypodermic needles

(needle-sticks), glass Pasteur pipettes, or broken glass.

3. Needle-stick injuries can be reduced by: (a) minimizing the use of syringes and

needles (e.g. simple devices are available for opening septum-stoppered bottles so

12. LABORATORY TECHNIQUES

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LABORATORY BIOSAFETY MANUAL

that pipettes can be used instead of syringes and needles; or (b) using engineered

sharp safety devices when syringes and needles are necessary.

4. Needles should never be recapped. Disposable articles should be discarded into

puncture-proof/puncture-resistant containers fitted with covers.

5. Plastic Pasteur pipettes should replace those made of glass.

Separation of serum

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 suitable disinfectant. They

should remain in the disinfectant for the appropriate time 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. Suitable disinfectants should be available for clean-up of splashes and spillages

(see Chapter 14).

Use of centrifuges

1. Satisfactory mechanical performance is a prerequisite of microbiological safety in

the use of laboratory centrifuges.

2. Centrifuges should be operated according to the manufacturer’s instructions.

3. Centrifuges should be placed at such a level that workers 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 manufacturer’s 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 in 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.

• 73 •

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 airflow if the

centrifuge is placed in a traditional open-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

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.

Use of tissue grinders

1. Glass grinders should be held in 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

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.

12. LABORATORY TECHNIQUES

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LABORATORY BIOSAFETY MANUAL

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 freezer’s 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. First decontaminate the outer surface of the ampoule.

2. Make a file mark on the tube near to the middle of the cotton or cellulose plug, if

present.

3. Hold the ampoule in alcohol-soaked cotton to protect hands before breaking it at

a file scratch.

4. Remove the top gently and treat as contaminated material.

5. If the plug is still above the contents of the ampoule, remove it with sterile forceps.

6. Add liquid for resuspension slowly to the ampoule to avoid frothing.

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.

Standard precautions with blood and other body fluids, tissues and excreta

Standard precautions (which include “universal precautions” (19)) are designed to

reduce the risk of transmission of microorganisms from both recognized and

unrecognized sources of infection (2).

Collection, labelling and transport of specimens

1. Standard precautions (2) 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

• 75 •

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 15 for transport requirements) and within the laboratory facility (see

section on Transport of specimens within the facility in this chapter). Request forms

should be placed in separate waterproof bags or envelopes.

5. Reception staff should not open these bags.

Opening specimen tubes and sampling contents

1. Specimen tubes should be opened in a biological safety cabinet.

2. Gloves must be worn. Eye and mucous membrane protection is also recommended

(goggles or face shields).

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 (see also section on Avoiding

injection of infectious materials in this chapter).

Films and smears for microscopy

Fixing and staining of blood, sputum and faecal samples for microscopy do 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.

3. Equipment should be disinfected at the end of each session, following manu-

facturers’ instructions.

Tissues

1. Formalin fixatives should be used.

2. Frozen sectioning should be avoided. When necessary, the cryostat should be

shielded and the operator should wear a safety face shield. For decontamination,

the temperature of the instrument should be raised to at least 20 °C.

12. LABORATORY TECHNIQUES

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LABORATORY BIOSAFETY MANUAL

Decontamination

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 5 g/l for blood spillages. Glutaraldehyde may be used for decontami-

nating surfaces (see Chapter 14).

Precautions with materials that may contain prions

Prions (also referred to as “slow viruses”) are associated with the transmissible spongi-

form encephalopathies (TSEs), notably Creutzfeldt-Jakob disease (CJD; including the

new variant form), Gerstmann-Sträussler-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 nature of the agent and 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. Recent studies indicate that prions in lingual and skeletal muscle

tissue may also present a potential infection risk (20–23).

As complete inactivation of prions is difficult to achieve, it is important to stress

the use of disposable instruments whenever possible, and to use a disposable protective

covering for the work surface of the biological safety cabinet.

The main precaution to be taken is to avoid ingestion of contaminated materials or

puncture of the laboratory worker’s skin. The following additional precautions should

be taken, as the agents are not killed by the normal processes of laboratory disinfection

and sterilization.

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 (plastic) should be used instead.

5. All manipulations must be conducted in biological safety cabinets.

6. Great care should be exercised to avoid aerosol production, ingestion, and cuts

and punctures of the skin.

• 77 •

7. Formalin-fixed tissues should be regarded as still infectious, even after prolonged

exposure to formalin.

8. Histological samples containing prions are substantially inactivated after exposure

to 96% formic acid for 1 h (24), (25).

9. Bench waste, including disposable gloves, gowns and aprons, should be autoclaved

using a porous load steam sterilizer at 134–137 °C for a single cycle of 18 min, or

six successive cycles of 3 min each, followed by incineration.

10. Non-disposable instruments, including steel mesh gloves, must be collected for

decontamination.

11. Infectious liquid waste contaminated with prions should be treated with sodium

hypochlorite containing available chlorine at 20 g/l (2%) (final concentration) for

1h.

12. 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.

13. Prion-contaminated biological safety cabinets and other surfaces can be

decontaminated with sodium hypochlorite containing available chlorine at 20 g/l

(2%) for 1 h.

14. High-efficiency particulate air (HEPA) filters should be incinerated at a minimum

temperature of 1000 °C after removal. Recommended additional steps prior to

incineration include:

a. spraying of the exposed face of the filter with lacquer hairspray prior to removal,

b. “bagging” of filters during removal, and

c. removal of the HEPA filter from the working chamber so that the inaccessible

plenum of the cabinet is not contaminated.

15. Instruments should be soaked in sodium hypochlorite containing available chlorine

at 20 g/l (2%) for 1 h and then rinsed well in water before autoclaving.

16. Instruments that cannot be autoclaved can be cleaned by repeated wetting with

sodium hypochlorite containing available chlorine at 20 g/l (2%) over a 1-h period.

Appropriate washing to remove residual sodium hypochlorite is required.

For further information on the handling of unconventional agents see references (12),

(26) and (27).

12. LABORATORY TECHNIQUES

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.

Contingency plan

The contingency plan should provide operational procedures for:

1. Precautions against natural disasters, e.g. fire, flood, earthquake and explosion

2. Biohazard risk assessment

3. Incident-exposure management and decontamination

4. Emergency evacuation of people and animals from the premises

5. Emergency medical treatment of exposed and injured persons

6. Medical surveillance of exposed persons

7. Clinical management of exposed persons

8. Epidemiological investigation

9. Post-incident continuation of operations.

In the development of this plan the following items should be considered for inclusion:

1. Identification of high-risk organisms

2. Location of high-risk areas, e.g. laboratories, storage areas, animal facilities

3. Identification of at-risk personnel and populations

4. 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

5. Lists of treatment and isolation facilities that can receive exposed or infected persons

6. Transport of exposed or infected persons

7. Lists of sources of immune serum, vaccines, drugs, special equipment and supplies

8. Provision of emergency equipment, e.g. protective clothing, disinfectants, chemical

and biological spill kits, decontamination equipment and supplies.

• 78 •

Emergency procedures for microbiological laboratories

Puncture wounds, cuts and abrasions

The affected individual should remove protective clothing, wash the hands and any

affected area(s), apply an appropriate skin disinfectant, and seek medical attention as

necessary. The cause of the wound and the organisms involved should be reported,

and appropriate and complete medical records kept.

Ingestion of potentially infectious material

Protective clothing should be removed and medical attention sought. Identification

of the material ingested and circumstances of the incident should be reported, and

appropriate and complete medical records kept.

Potentially infectious aerosol release (outside 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 an appropriate amount

of time (e.g. 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

(e.g. for 24 h).

Signs 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.

Broken containers and spilled infectious substances

Broken containers contaminated with infectious substances and spilled infectious

substances should be covered with a cloth or paper towels. Disinfectant should then

be poured over these and left for the appropriate amount of time. The cloth or paper

towels and the broken material can 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. 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 infectious material in centrifuges

not having sealable buckets

If a breakage occurs or is suspected while the machine is running, the motor should be

switched off and the machine left closed (e.g. for 30 min) to allow settling. If a breakage

is discovered after the machine has stopped, the lid should be replaced immediately

13. CONTINGENCY PLANS AND EMERGENCY PROCEDURES

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LABORATORY BIOSAFETY MANUAL

and left closed (e.g. 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 a noncorrosive disinfectant known to be active against the organisms concerned

(see Chapter 14). Unbroken, capped tubes may be placed in disinfectant in a separate

container and recovered.

The centrifuge bowl should be swabbed with the same disinfectant, at the appropriate

dilution, 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 breakage is suspected within the safety cup, the safety cap should be loosened

and the bucket autoclaved. Alternatively, the safety cup may be chemically disinfected.

Fire and natural disasters

Fire and other services should be involved in the development of emergency

preparedness 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.

After a natural disaster, 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. Infectious materials should be

collected in leakproof boxes or strong disposable bags.

Salvage or final disposal should be determined by biosafety staff on the basis of

local ordinances.

Emergency services: whom to contact

The telephone numbers and addresses of the following should be prominently displayed

in the facility:

1. The institution or laboratory itself (the address and location may not be known in

detail by the caller or the services called)

2. Director of the institution or laboratory

3. Laboratory supervisor

4. Biosafety officer

5. Fire services

6. Hospitals/ambulance services/medical staff (names of individual clinics,

departments, and/or medical staff, if possible)

• 81 •

7. Police

8. Medical officer

9. Responsible technician

10. Water, gas and electricity services.

Emergency equipment

The following emergency equipment must be available:

1. First-aid kit, including universal and special antidotes

2. Appropriate fire extinguishers, fire blankets

The following are also suggested but may be varied according to local circumstances:

1. Full protective clothing (one-piece coveralls, gloves and head covering – for

incidents involving microorganisms in Risk Groups 3 and 4)

2. Full-face respirators with appropriate chemical and particulate filter canisters

3. Room disinfection apparatus, e.g. sprays and formaldehyde vaporizers

4. Stretcher

5. Tools, e.g. hammers, axes, spanners, screwdrivers, ladders, ropes

6. Hazard area demarcation equipment and notices.

For further information see references (12) and (28).

13. CONTINGENCY PLANS AND EMERGENCY PROCEDURES

14. Disinfection and sterilization

A basic knowledge of disinfection and sterilization is crucial for biosafety in the

laboratory. Since heavily soiled items cannot promptly be disinfected or sterilized, it is

equally important to understand the fundamentals of cleaning prior to disinfection

(precleaning). In this regard, the following general principles apply to all known classes

of microbial pathogens.

Specific decontamination requirements will depend on the type of experimental

work and the nature of the infectious agent(s) being handled. The generic information

given here can be used to develop both standardized and more specific procedures to

deal with biohazard(s) involved in a particular laboratory.

Contact times for disinfectants are specific for each material and manufacturer.

Therefore, all recommendations for use of disinfectants should follow manufacturers’

specifications.

Definitions

Many different terms are used for disinfection and sterilization. The following are

among the more common in biosafety:

Antimicrobial – An agent that kills microorganisms or suppresses their growth and

multiplication.

Antiseptic – A substance that inhibits the growth and development of microorganisms

without necessarily killing them. Antiseptics are usually applied to body surfaces.

Biocide – A general term for any agent that kills organisms.

Chemical germicide – A chemical or a mixture of chemicals used to kill micro-

organisms.

Decontamination – Any process for removing and/or killing microorganisms. The

same term is also used for removing or neutralizing hazardous chemicals and radio-

active materials.

Disinfectant – A chemical or mixture of chemicals used to kill microorganisms, but

not necessarily spores. Disinfectants are usually applied to inanimate surfaces or

objects.

Disinfection – A physical or chemical means of killing microorganisms, but not

necessarily spores.

Microbicide – A chemical or mixture of chemicals that kills microorganisms. The

term is often used in place of “biocide”, “chemical germicide” or “antimicrobial”.

• 82 •

Sporocide – A chemical or mixture of chemicals used to kill microorganisms and

spores.

Sterilization – A process that kills and/or removes all classes of microorganisms and

spores.

Cleaning laboratory materials

Cleaning is the removal of dirt, organic matter and stains. Cleaning includes brushing,

vacuuming, dry dusting, washing or damp mopping with water containing a soap or

detergent. Dirt, soil and organic matter can shield microorganisms and can interfere

with the killing action of decontaminants (antiseptics, chemical germicides and

disinfectants).

Precleaning is essential to achieve proper disinfection or sterilization. Many germi-

cidal products claim activity only on precleaned items. Precleaning must be carried

out with care to avoid exposure to infectious agents.

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.

Chemical germicides

Many types of chemicals can be used as disinfectants and/or antiseptics. As there is an

ever-increasing number and variety of commercial products, formulations must be

carefully selected for specific needs.

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. 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 or the environment. They should be

selected, stored, handled, used and disposed of with care, following manufacturers’

instructions. For personal safety, gloves, aprons and eye protection are recommended

when preparing dilutions of chemical germicides.

Chemical germicides are generally not required for regular cleaning of floors, walls,

equipment and furniture. However, their use may be appropriate in certain cases of

outbreak control.

Proper use of chemical germicides 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 for economic reasons, inventory control and to limit

environmental pollution.

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 12 summarizes the

recommended dilutions of chlorine-releasing compounds.

14. DISINFECTION AND STERILIZATION

• 83 •

• 84 •

LABORATORY BIOSAFETY MANUAL

Chlorine (sodium hypochlorite)

Chlorine, a fast-acting oxidant, is a widely available and broad-spectrum chemical

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 recom-

mended for dealing with biohazardous spillage and in the presence of large amounts

of organic matter. 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 concen-

trations 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.

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

Table 12. Recommended dilutions of chlorine-releasing compounds

“CLEAN” CONDITIONSa“DIRTY” CONDITIONSb

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

Sodium dichloroisocyanurate powder 1.7 g/l 8.5 g/l

(60% available chlorine)

Sodium dichloroisocyanurate tablets 1 tablet 4 tablets

(1.5 g available chlorine per tablet) per litre per litre

Chloramine (25% available chlorine)c20 g/l 20 g/l

aAfter removal of bulk material.

bFor flooding, e.g. on blood or before removal of bulk material.

cSee text.

• 85 •

14. DISINFECTION AND STERILIZATION

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 1–2 mg/l

available chlorine.

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 to prevent the rapid

release of chlorine gas. Many by-products of chlorine can be harmful to humans and

the environment, so that indiscriminate use of chlorine-based disinfectants, in

particular bleach, should be avoided.

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

1g/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 l of

water will give approximately 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.

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.

Chlorine dioxide

Chlorine dioxide (ClO2) is a strong and fast-acting germicide, disinfectant agent and

oxidizer, often reported to be active at concentrations levels lower than those needed

by chlorine as bleach. Chlorine dioxide is unstable as a gas and will undergo

decomposition into chlorine gas (Cl2), oxygen gas (O2), giving off heat. However,

chlorine dioxide is soluble in water and stable in an aqueous solution. Chlorine dioxide

can be obtained in two ways: (1) on-site generation by mixing of two separate

components, hydrochloric acid (HCl) and sodium chlorite (NaClO2); and (2) ordering

its stabilized form, which is then activated on-site when required.

Of the oxidizing biocides, chlorine dioxide is the most selective oxidant. Ozone and

chlorine are much more reactive than chlorine dioxide, and they will be consumed by

most organic compounds. Chlorine dioxide, however, reacts only with reduced sulfur

• 86 •

LABORATORY BIOSAFETY MANUAL

compounds, secondary and tertiary amines, and some other highly reduced and reactive

organic compounds. A more stable residue can therefore be achieved with chlorine

dioxide at much lower doses than when using either chlorine or ozone. Generated

properly, chlorine dioxide can be used more effectively than ozone or chlorine in cases

of higher organic loading because of its selectivity.

Formaldehyde

Formaldehyde (HCHO) is a gas that kills all microorganisms and spores at temperatures

above 20 °C. However, it is not active against prions.

Formaldehyde 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 in this chapter).

Formaldehyde (5% formalin in water) may be used as a liquid disinfectant.

Formaldehyde is a suspected carcinogen. It is a dangerous, irritant gas that 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 area. National chemical safety

regulations must be followed.

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.

Glutaraldehyde is generally supplied as a solution with a concentration of about

20 g/l (2%) and some products may 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 1–4 weeks depending on the formulation and type and

frequency of its use. Dipsticks supplied 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.

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. National chemical safety regulations must be followed.

Phenolic compounds

Phenolic compounds, a broad group of agents, were among the earliest germicides.

However, more recent safety concerns restrict their use. They are active against veg-

etative bacteria and lipid-containing viruses and, when properly formulated, also show

• 87 •

activity against mycobacteria. They are not active against spores and their activity

against nonlipid viruses is variable. Many phenolic products are used for the decon-

tamination 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.

Some phenolic compounds are sensitive to and may be inactivated by water hardness

and therefore must be diluted with distilled or deionized water.

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. National chemical safety regulations must be followed.

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

some vegetative bacteria and lipid-containing viruses. Certain types (e.g. benzalkonium

chloride) are used as antiseptics.

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.

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. 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

14. DISINFECTION AND STERILIZATION

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LABORATORY BIOSAFETY MANUAL

that ethanol is ineffective against spores and may not kill all types of nonlipid viruses.

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. Bottles with alcohol-containing solutions must be clearly

labelled to avoid 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. Polyvidone-iodine 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.

Iodine can be toxic. Organic iodine-based products must be stored at 4–10 °C to

avoid the growth of potentially harmful bacteria in them.

Hydrogen peroxide and peracids

Like chlorine, hydrogen peroxide (H2O2) and peracids are strong oxidants and can be

potent broad-spectrum 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 5–10 times its volume with sterilized water. However,

such 3–6% 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.

Hydrogen peroxide and peracids can be corrosive to metals such as aluminium,

copper, brass and zinc, and can also decolorize 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.

Local environmental decontamination

Decontamination of the laboratory space, its furniture and its equipment requires a

combination of liquid and gaseous disinfectants. Surfaces can be decontaminated using

• 89 •

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. This is a highly

dangerous process that requires specially trained personnel. 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 in this chapter.)

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 smaller spaces with hydrogen peroxide vapour is also effective but

requires specialized equipment to generate the vapour.

Decontamination of biological safety cabinets

To decontaminate Class I and Class II cabinets, equipment that independently generates,

circulates and neutralizes formaldehyde gas is available. Alternatively, the appropriate

amount of paraformaldehyde (final concentration of 0.8% paraformaldehyde in air)

should be placed in a frying pan on an electric hot plate. Another frying pan, containing

10% more ammonium bicarbonate than paraformaldehyde, on a second hot plate is

also placed inside the cabinet. The hot plate leads are plugged in outside the cabinet,

so that operation of the pans can be controlled from the outside by plugging and

unplugging the hot plates as necessary. 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). Heavy gauge plastic sheeting is

taped over the front opening and exhaust port to make sure that the gas cannot seep

into the room. Penetration of the electric leads passing through the front closure must

also be sealed with duct tape.

The plate for the paraformaldehyde pan is plugged in. It is unplugged when all the

paraformaldehyde has vaporized. The cabinet is left undisturbed for at least 6 h. The

plate for the second pan is then plugged in and the ammonium bicarbonate is allowed

to vaporize. This plate is then unplugged and the cabinet blower is switched on for

two intervals of approximately 2 s each to allow the ammonium bicarbonate gas to

circulate. The cabinet should be left undisturbed for 30 min before the front closure

(or plastic sheeting) and the exhaust port sheeting are removed. The cabinet surfaces

should be wiped down to remove residues before use.

14. DISINFECTION AND STERILIZATION

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LABORATORY BIOSAFETY MANUAL

Hand-washing/hand decontamination

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 before leaving the laboratory.

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 may be used).

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 may be used to decontaminate lightly

soiled hands when proper hand-washing is not available.

Heat disinfection and sterilization

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 2–4 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.

Autoclaving

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:

1. 3 min holding time at 134 °C

2. 10 min holding time at 126 °C

3. 15 min holding time at 121 °C

4. 25 min holding time at 115 °C.

Examples of different autoclaves include the following.

Gravity displacement autoclaves. Figure 10 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.

• 91 •

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 displace-

ment 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.

Loading autoclaves

Materials should be loosely packed in the chamber for easy steam penetration and air

removal. Bags should allow the steam to reach their contents.

14. DISINFECTION AND STERILIZATION

Figure 10. Gravity displacement autoclave

pressure

gauge

baffle

to jacket

valve

steam supply

to chamber

Valve

to vacuum pump

or steam ejector

near-to-steam traps

strainers

non-return valve

door

cotton wool

filter

valve

safety

valve

combined pressure

and vacuum gauge

safety

valve

chamber

valve

Jacket

WHO 02.139

• 92 •

LABORATORY BIOSAFETY MANUAL

Precautions in the use of autoclaves

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.

2. A preventive maintenance programme should include regular inspection of the

chamber, door seals and all gauges and controls by qualified personnel.

3. The steam should be saturated and free from chemicals (e.g. corrosion inhibitors)

that could contaminate the items being sterilized.

4. All materials to be autoclaved should be in containers that allow ready removal of

air and permit good heat penetration; the chamber should be loosely packed so

that steam will reach the load evenly.

5. 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.

6. Slow exhaust settings should be used when autoclaving liquids, as they may boil

over when removed due to superheating.

7. Operators should wear suitable gloves and visors for protection when opening the

autoclave, even when the temperature has fallen below 80 °C.

8. 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.

9. The drain screen filter of the chamber (if available) should be removed and cleaned

daily.

10. Care should be taken to ensure that the relief valves of pressure cooker autoclaves

do not become blocked by paper, etc. in the load.

Incineration

Incineration is useful for disposing of animal carcasses as well as anatomical and other

laboratory waste, with or without prior decontamination (see Chapter 3). Incineration

of infectious materials is an alternative to autoclaving only if the incinerator is under

laboratory control.

Proper incineration requires 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

• 93 •

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 ongoing concerns regarding the possible negative environmental effects

of existing or proposed incinerators, and efforts continue to make incinerators more

environmentally friendly and energy-efficient.

Disposal

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

Chapter 3).

For further information see references (13) and (29–39).

14. DISINFECTION AND STERILIZATION

15. Introduction to the transport

of infectious substances

Transport of infectious and potentially infectious materials is subject to strict national

and international regulations. These regulations describe the proper use of packaging

materials, as well as other shipping requirements.

Laboratory personnel must ship infectious substances according to applicable

transport regulations. Compliance with the rules will:

1. Reduce the likelihood that packages will be damaged and leak, and thereby

2. Reduce the exposures resulting in possible infections

3. Improve the efficiency of package delivery.

International transport regulations

The regulations for the transport of infectious materials (by any mode of transport)

are based upon the United Nations Model Regulations on the Transport of Dangerous

Goods (40). These recommendations are developed by the United Nations Committee

of Experts on the Transport of Dangerous Goods (UNCETDG). To become legally

binding, the United Nations Model Regulations have to be introduced into national

regulations and international modal regulations by the competent authorities (e.g.

the Technical Instructions for the Safe Transport of Dangerous Goods by Air (41) of the

International Civil Aviation Organization (ICAO) for air transport and the European

Agreement concerning the International Carriage of Dangerous Goods by Road (ADR)

(42).

The International Air Transport Association (IATA) issues Infectious Substances

Shipping Guidelines (43) every year. IATA guidelines must follow ICAO’s Technical

Instructions as a minimal standard, but may impose additional restrictions. IATA

guidelines must be followed if a shipment is carried by members of IATA.

Since the United Nations Model Regulations on the Transport of Dangerous Goods is

a dynamic set of recommendations subject to biennial amendments, the reader is

referred to the latest issuances of national and international modal regulations for

applicable regulatory texts.

WHO serves in an advisory capacity to UNCETDG. Major changes to the transport

regulations pertaining to the transport of infectious substances were introduced into

the 13th edition (2003) of the United Nations Model Regulations (40). Guidance on

the background to adopted amendments is available from WHO (44).

• 94 •

International modal regulations are not intended to supersede any local or national

requirements. However, in situations where national requirements do not exist,

international modal regulations should be followed.

It is important to note that international transport of infectious substances is also

dependent on and subject to national import/export regulations.

The basic triple packaging system

The triple packaging system, the choice for the transport of infectious and potentially

infectious substances, is exemplified in Figure 11. This packaging system consists of

three layers: the primary receptacle, the secondary packaging and the outer packaging.

The primary receptacle containing the specimen must be watertight, leakproof and

appropriately labelled as to content. The primary receptacle is wrapped in enough

absorbent material to absorb all fluid in case of breakage or leakage.

A second watertight, leakproof packaging is used to enclose and protect the primary

receptacle(s). Several wrapped primary receptacles may be placed in a single secondary

packaging. Volume and/or weight limits for packaged infectious substances are included

in certain regulatory texts.

The third layer protects the secondary packaging from physical damage while in

transit. Specimen data forms, letters and other types of information that identify or

describe the specimen and identify the shipper and receiver, and any other

documentation required, must also be provided according to latest regulations.

The United Nations Model Regulations prescribe the use of two different triple

packaging systems. The basic triple packaging system applies for the transport of a

variety of infectious substances; however, high-risk organisms must be shipped

according to more stringent requirements. For further details about the use of the

different packagings according to the materials to be transported, it is advisable to

consult national and/or international modal regulations for applicable regulatory texts.

Spill clean-up procedure

In the event of a spill of infectious or potentially infectious material, the following

spill clean-up procedure should be used.

1. Wear gloves and protective clothing, including face and eye protection if indicated.

2. Cover the spill with cloth or paper towels to contain it.

3. Pour an appropriate disinfectant over the paper towels and the immediately

surrounding area (generally, 5% bleach solutions are appropriate; but for spills on

aircraft, quaternary ammonium disinfectants should be used).

4. Apply disinfectant concentrically beginning at the outer margin of the spill area,

working toward the centre.

5. After the appropriate amount of time (e.g. 30 min), clear away the materials. If

there is broken glass or other sharps involved, use a dustpan or a piece of stiff

cardboard to collect the material and deposit it into a puncture-resistant container

for disposal.

15. INTRODUCTION TO THE TRANSPORT OF INFECTIOUS SUBSTANCES

• 95 •

• 96 •

LABORATORY BIOSAFETY MANUAL

Figure 11. Examples of triple packaging systems

(graphics kindly provided by IATA, Montreal, Canada)

Packing and labelling of Category A infectious substances

Packing and labelling of Category B infectious substances

• 97 •

6. Clean and disinfect the area of the spillage (if necessary, repeat steps 2–5).

7. Dispose of contaminated materials into a leakproof, puncture-resistant waste

disposal container.

8. After successful disinfection, inform the competent authority that the site has now

been decontaminated

15. INTRODUCTION TO THE TRANSPORT OF INFECTIOUS SUBSTANCES

PART V

Introduction to

biotechnology

16. Biosafety and recombinant

DNA technology

• 101 •

Recombinant DNA technology involves combining genetic material 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 that could

represent a biohazard if they escaped from the laboratory. This concern became the

focus of a scientific conference held in Asilomar, CA, USA, in 1975 (45). At that meeting,

safety issues were discussed and the first guidelines for recombinant DNA technology

were proposed. The subsequent 25+ years of research experience have demonstrated

that genetic engineering may be conducted in a safe manner when an appropriate risk

assessment is performed and adequate safety measures are used.

Recombinant DNA technology or genetic engineering was first used to clone DNA

segments in bacterial hosts in order to overexpress specific gene products for further

studies. Recombinant DNA molecules have also been used to create GMOs such as

transgenic and “knock-out” animals and transgenic plants.

Recombinant DNA technology has already had an enormous impact on biology

and medicine, and will probably have an even greater influence now that the nucleotide

sequence of the entire human genome is available. Tens of thousands of genes of yet

unknown functions will be studied using recombinant DNA technology. Gene therapy

may become a routine treatment for certain diseases, and new vectors for gene transfer

are likely to be devised using genetic engineering techniques. Also, transgenic plants

produced by recombinant DNA technology may play an increasingly important role

in modern agriculture.

Experiments involving the construction or use of GMOs should be conducted after

performing a biosafety risk assessment. The pathogenic properties and any potential

hazards associated with such organisms may be novel and not well-characterized. 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 should be evaluated. These factors should help determine the biosafety

level that is required for the safe handling of the resulting GMO, and identify the

biological and physical containment systems that should be used.

• 102 •

LABORATORY BIOSAFETY MANUAL

Biosafety considerations for biological expression systems

Biological expression systems consist of vectors and host cells. A number of criteria

must be satisfied to make them effective and safe to use. An example of such a biological

expression system is plasmid pUC18. Frequently used as a cloning vector in

combination with Escherichia coli K12 cells, the pUC18 plasmid has been entirely

sequenced. All genes required for expression in other bacteria have been deleted from

its precursor plasmid pBR322. E. coli K12 is a non-pathogenic strain that cannot

permanently colonize the gut of healthy humans or animals. Routine genetic

engineering experiments can safely be performed in E. coli K12/pUC18 at Biosafety

Level 1, provided the inserted foreign DNA expression products do not require higher

biosafety levels.

Biosafety considerations for expression vectors

Higher biosafety levels may be required when:

1. The expression of DNA sequences derived from pathogenic organisms may increase

the virulence of the GMO

2. Inserted DNA sequences are not well characterized, e.g. during preparation of

genomic DNA libraries from pathogenic microorganisms

3. Gene products have potential pharmacological activity

4. Gene products code for toxins.

Viral vectors for gene transfer

Viral vectors, e.g. adenovirus vectors, are used for the transfer of genes to other cells.

Such vectors lack certain virus replication genes and are propagated in cell lines that

complement the defect.

Stocks of such vectors may be contaminated with replication-competent viruses,

generated by rare spontaneous recombination events in the propagating cell lines, or

may derive from insufficient purification. These vectors should be handled at the same

biosafety level as the parent adenovirus from which they are derived.

Transgenic and “knock-out” animals

Animals carrying foreign genetic material (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

• 103 •

receptor generated in different laboratories were susceptible to poliovirus infection

by various inoculation routes and the resulting disease was clinically and histo-

pathologically 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 many parts of the world.

The discussions focus on the food-safety of such plants, and on the long-term ecological

consequences of their cultivation.

Transgenic plants expressing genes of animal or human origin are used to develop

medicinal and nutritional products. A risk assessment should determine the appropriate

biosafety level for the production of these plants.

Risk assessments for genetically modified organisms

Risk assessments for work with GMOs should consider the characteristics of donor

and recipient/host organisms.

Examples of characteristics for consideration include the following.

Hazards arising directly from the inserted gene (donor organism)

Assessment is necessary in situations where the product of the inserted gene has known

biologically or pharmacologically active properties that may give rise to harm, for

example:

1. Toxins

2. Cytokines

3. Hormones

4. Gene expression regulators

5. Virulence factors or enhancers

6. Oncogenic gene sequences

7. Antibiotic resistance

8. Allergens.

The consideration of such cases should include an estimation of the level of expression

required to achieve biological or pharmacological activity.

16. BIOSAFETY AND RECOMBINANT DNA TECHNOLOGY

Hazards associated with the recipient/host

1. Susceptibility of the host

2. Pathogenicity of the host strain, including virulence, infectivity and toxin

production

3. Modification of the host range

4. Recipient immune status

5. Consequences of exposure.

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).

1. Is there an increase in infectivity or pathogenicity?

2. Could any disabling mutation within the recipient be overcome as a result of the

insertion of the foreign gene?

3. Does the foreign gene encode a pathogenicity determinant from another organism?

4. If the foreign DNA does include a pathogenicity determinant, is it foreseeable that

this gene could contribute to the pathogenicity of the GMO?

5. Is treatment available?

6. Will the susceptibility of the GMO to antibiotics or other forms of therapy be

affected as a consequence of the genetic modification?

7. Is eradication of the GMO achievable?

Further considerations

The use of whole animals or plants for experimental purposes also requires careful

consideration. Investigators must comply with the regulations, restrictions and

requirements for the conduct of work with GMOs in host countries and institutions.

Countries may have national authorities that establish guidelines for work with

GMOs, and may 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 a dynamic process that takes into account new developments

and the progress of science. The performance of appropriate risk assessments will

assure that the benefits of recombinant DNA technology remain available to

humankind in the years to come.

For further information see references (17) and (46–48).

• 104 •

LABORATORY BIOSAFETY MANUAL

PART VI

Chemical, fire and

electrical safety

17. Hazardous chemicals

• 107 •

Workers in microbiological laboratories are not only exposed to pathogenic

microorganisms, but also to chemical hazards. It is important that they have proper

knowledge of the toxic effects of these chemicals, the routes of exposure and the hazards

that may be associated with handling and storage (see Annex 5). Material safety data

sheets or other chemical hazard information are available from chemical manufacturers

and/or suppliers. These should be accessible in laboratories where these chemicals are

used, e.g. as part of a safety or operations manual.

Routes of exposure

Exposure to hazardous chemicals may occur by:

1. Inhalation

2. Contact

3. Ingestion

4. Needle-sticks

5. Through broken skin.

Storage of chemicals

Only amounts of chemicals necessary for daily use should be stored in the laboratory.

Bulk stocks should be kept in specially designated rooms or buildings.

Chemicals should not be stored in alphabetical order.

General rules regarding chemical incompatibilities

To avoid fire and/or explosions, substances in the left-hand column of Table 13 should

be stored and handled so that they cannot come into contact with the corresponding

substances in the right-hand column of the table.

Toxic effects of chemicals

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 and tissues may be adversely affected or seriously

damaged. Some chemicals are known to be carcinogenic or teratogenic.

• 108 •

LABORATORY BIOSAFETY MANUAL

Table 13. General rules for chemical incompatibilities

SUBSTANCE CATEGORY INCOMPATIBLE SUBSTANCES

Alkali metals, e.g. sodium, potassium, Carbon dioxide, chlorinated hydrocarbons, water

caesium and lithium

Halogens Ammonia, acetylene, hydrocarbons

Acetic acid, hydrogen sulfide, aniline, Oxidizing agents, e.g. chromic acid, nitric acid,

hydrocarbons, sulfuric acid peroxides, permanganates

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.

For detailed information on the toxic effects of chemicals see Annex 5.

Explosive chemicals

Azides, often used in antibacterial solutions, should not be allowed to come into contact

with copper or lead (e.g. in waste pipes and plumbing), as they may explode violently

when subjected even to a mild impact.

Ethers that have aged and dried to crystals are extremely unstable, and potentially

explosive.

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 spills

Most manufacturers of laboratory chemicals issue charts describing methods for

dealing with spills. 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:

1. Chemical spill kits

2. Protective clothing, e.g. heavy-duty rubber gloves, overshoes or rubber boots,

respirators

3. Scoops and dustpans

4. Forceps for picking up broken glass

5. Mops, cloths and paper towels

6. Buckets

• 109 •

7. Soda ash (sodium carbonate, Na2CO3) or sodium bicarbonate (NaHCO3) for

neutralizing acids and corrosive chemicals

8. Sand (to cover alkali spills)

9. Non-flammable detergent.

The following actions should be taken in the event of a significant chemical spill.

1. Notify the appropriate safety officer.

2. Evacuate non-essential personnel from the area.

3. Attend to persons who may have been contaminated.

4. If the spilled material is flammable, extinguish all open flames, turn off gas in the

room and adjacent areas, open windows (if possible), and switch off electrical

equipment that may spark.

5. Avoid breathing vapour from spilled material.

6. Establish exhaust ventilation if it is safe to do so.

7. Secure the necessary items (see above) to clean up the spill.

Compressed and liquefied gases

Information regarding storage of compressed and liquefied gases is given in Table 14.

Table 14. Storage of compressed and liquefied gases

CONTAINER STORAGE INFORMATION

Compressed gas cylinders and • Should be securely fixed (e.g. chained) to the wall

liquefied gas containersa,b or a solid bench so that they are not inadvertently

dislodged.

•Must be transported with their caps in place and

supported on trolleys.

•Should be stored in bulk in an appropriate facility at

some distance from the laboratory. This area should

be locked and appropriately identified.

•Should not be placed near radiators, open flames

other heat sources, sparking electrical equipment,

or in direct sunlight.

Small, single-use gas cylindersa,b •Must not be incinerated.

aThe main high-pressure valve should be turned off when the equipment is not in use and when the room

is unoccupied.

bRooms where flammable gas cylinders are used and/or stored should be identified by warning notices on

the doors.

For further information see references (1) and (49–51), and Annex 5.

17. HAZARDOUS CHEMICALS

18. Additional laboratory hazards

Laboratory personnel may confront hazards posed by forms of energy including fire,

electricity, radiation and noise. Basic information about each of these is presented in

this chapter.

Fire hazards

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 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:

1. Electrical circuit overloading

2. Poor electrical maintenance, e.g. poor and perished insulation on cables

3. Excessively long gas tubing or long electrical leads

4. Equipment unnecessarily left switched on

5. Equipment that was not designed for a laboratory environment

6. Open flames

7. Deteriorated gas tubing

8. Improper handling and storage of flammable or explosive materials

9. Improper segregation of incompatible chemicals

10. Sparking equipment near flammable substances and vapours

11. Improper or inadequate ventilation.

Fire-fighting equipment should be placed near room doors and at strategic points in

corridors and hallways. This equipment may include hoses, buckets (of water or sand)

and a fire extinguisher. Fire extinguishers should be regularly inspected and maintained,

and their shelf-life kept up to date. Specific types and uses of fire extinguishers are

shown in Table 15.

• 110 •

Table 15. Types and uses of fire extinguishers

TYPE USE FOR DO NOT USE FOR

Water Paper, wood, fabric Electrical fires, flammable liquids,

burning metals

Carbon dioxide (CO2)Flammable liquids and gases, Alkali metals, paper

extinguisher gases electrical fires

Dry powder Flammable liquids and gases, Reusable equipment and

alkali metals, electrical fires instruments, as residues are very

difficult to remove

Foam Flammable liquids Electrical fires

For further information see reference (49).

Electrical hazards

It is essential that all electrical installations and equipment are inspected and tested

regularly, including earthing/grounding systems.

Circuit-breakers and earth-fault-interrupters should be installed in appropriate

laboratory electrical circuits. Circuit-breakers do not protect people; they are intended

to protect wiring from being overloaded with electrical current and hence to prevent

fires. Earth-fault-interrupters are intended to protect people from electric shock.

All laboratory electrical equipment should be earthed/grounded, preferably through

three-prong plugs.

All laboratory electrical equipment and wiring should conform to national electrical

safety standards and codes.

Noise

The effect of excessive noise is insidious over time. Some types of laboratory equipment,

such as certain laser systems, as well as facilities where animals are housed, can produce

significant noise exposure to workers. Noise measurement surveys can be conducted

to determine the noise hazard. Where warranted by data, engineering controls such as

enclosures or barriers around noisy equipment or between noisy areas and other work

areas, can be considered. Where noise levels cannot be abated and where laboratory

personnel routinely experience excessive exposures, a hearing conservation programme

that includes the use of hearing protection while working in hazardous noise and a

medical monitoring programme to determine the effect of noise on the workers should

be instituted.

Ionizing radiation

Radiological protection is concerned with protecting humans against the harmful

effects of ionizing radiation, which include:

18. ADDITIONAL LABORATORY HAZARDS

• 111 •

• 112 •

LABORATORY BIOSAFETY MANUAL

1. Somatic effects, e.g. clinical symptoms observable in exposed individuals. Somatic

effects include radiation-induced cancers, e.g. leukaemia and bone, lung and skin

cancers, the onset of which may occur many years after irradiation. Less severe

somatic effects include minor skin damage, hair loss, blood deficiencies, gastro-

intestinal damage and cataract formation.

2. Hereditary effects, e.g. symptoms observed in the descendants of exposed indi-

viduals. The hereditary effects of radiation exposure to the gonads include chromo-

some damage or gene mutation. Irradiation of the germ cells in the gonads in high

doses can also cause cell death, resulting in impaired fertility in both sexes or

menstrual changes in women. Exposure of the developing fetus, particularly in

weeks 8–15 of pregnancy, may increase the risk of congenital malformations, mental

impairment or radiation-induced cancers in later life.

Principles of ionizing radiation protection

To limit the harmful effects of ionizing radiation, the use of radioisotopes should be

controlled and should comply with relevant national standards. Protection from

radiation is managed on the basis of four principles:

1. Minimizing the time of radiation exposure

2. Maximizing the distance from the radiation source

3. Shielding the radiation source

4. Substituting the use of radionuclides with non-radiometric techniques.

Protection activities include the following.

1. Time. The time of exposure experienced during manipulations of radioactive

material can be reduced by:

—Practising new and unfamiliar techniques without using the radionuclide until

the techniques are mastered

—Working with radionuclides in a deliberate and timely manner without rushing

—Ensuring that all radioactive sources are returned to storage immediately after

use

—Removing radioactive waste from the laboratory at frequent intervals

—Spending as little time as possible in the radiation area or laboratory

—Exercising effective time management and planning of laboratory manipulations

involving radioactive material.

The less time spent in a radiation field, the smaller the received personal dose, as

described in the equation:

Dose = Dose rate

time

2. Distance. The dose rate for most γ- and X-radiation varies as the inverse square of

the distance from a point source:

Dose rate = Constant

1/Distance2

• 113 •

Doubling the distance from a radiation source will result in reducing the exposure

by one-fourth over the same period of time. Various devices and mechanical aids

are used to increase the distance between the operator and the radiation source,

e.g. long-handled tongs, forceps, clamps and remote pipetting aids. Note that a

small increase in distance can result in significant decrease in the dose rate.

3. Shielding. Radiation energy-absorbing or attenuating shields placed between the

source and the operator or other occupants of the laboratory will help limit their

exposure. The choice and thickness of any shielding material depends on the

penetrating ability (type and energy) of the radiation. A barrier of acrylic, wood

or lightweight metal, thickness 1.3–1.5 cm, provides shielding against high-energy

β particles, whereas high-density lead is needed to shield against high energy γ-

and X-radiation.

4. Substitution. Radionuclide-based materials should not be used when other

techniques are available. If substitution is not possible, then the radionuclide with

the least penetrating power or energy should be used.

Safe practices for work with radionuclides

Rules for working with radioactive substances should include considerations in four

areas:

1. Radiation area

2. Work-bench area

3. Radioactive waste area

4. Records and emergency response.

Some of the most important rules include the following:

1. Radiation area

—Use radioactive substances only in dedicated areas.

—Allow the presence of essential staff only.

—Use personal protective equipment, including laboratory coats, safety spectacles

and disposable gloves.

—Monitor personal radiation exposures.

Laboratories where radionuclides are used should be designed to simplify contain-

ment, cleaning and decontamination. The radionuclide work area should be located

in a small room adjoining the main laboratory, or in a dedicated area within the

laboratory away from other activities. Signs displaying the international radiation

hazard symbol should be posted at the entrance to the radiation area (Figure 12).

2. Work-bench area

—Use spill trays lined with disposable absorbent materials.

—Limit radionuclide quantities.

—Shield radiation sources in the radiation, work bench and radioactive waste

areas.

18. ADDITIONAL LABORATORY HAZARDS

—Mark radiation containers with the radiation symbol, including radionuclide

identity, activity and assay date.

—Use radiation meters to monitor working areas, protective clothing and hands

after completion of work.

—Use appropriately shielded transport containers.

3. Radioactive waste area

—Remove radioactive waste frequently from the working area.

—Maintain accurate records of use and disposal of radioactive materials.

—Screen dosimetry records for materials exceeding the dose limits.

—Establish and regularly exercise emergency response plans.

—In emergencies, assist injured persons first.

—Clean contaminated areas thoroughly.

—Request assistance from the safety office, if available.

—Write and keep incident reports.

Figure 12. International radiation

hazard symbol

• 114 •

LABORATORY BIOSAFETY MANUAL

PART VII

Safety organization

and training

19. The biosafety officer and

biosafety committee

• 117 •

It is essential that each laboratory organization has a comprehensive safety policy, a

safety manual, and supporting programmes for their implementation. The

responsibility for this normally rests with the director or head of the institute or

laboratory, who may delegate certain duties to a biosafety officer or other appropriate

personnel.

Laboratory safety is also the responsibility of all supervisors and laboratory

employees, and individual workers are responsible for their own safety and that of

their colleagues. Employees are expected to perform their work safely and should report

any unsafe acts, conditions or incidents to their supervisor. Periodic safety audits by

internal or external personnel are desirable.

Biosafety officer

Wherever possible a biosafety officer should be appointed to ensure that biosafety

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 biosafety, the person designated should possess the professional

competence necessary to suggest, review and approve specific activities that follow

appropriate biocontainment and biosafety procedures. The biosafety officer should

apply relevant national and international rules, regulations and guidelines, as well as

assist the laboratory in developing standard operating procedures. The person

appointed must have a technical background in microbiology, biochemistry and basic

physical and biological sciences. Knowledge of laboratory and clinical practices and

safety, including containment equipment, and engineering principles relevant to the

design, operation and maintenance of facilities is highly desirable. The biosafety officer

should also be able to communicate effectively with administrative, technical and

support personnel.

The activities of the biosafety officer should include the following:

1. Biosafety, biosecurity and technical compliance consultations.

2. Periodic internal biosafety audits on technical methods, procedures and protocols,

biological agents, materials and equipment.

• 118 •

LABORATORY BIOSAFETY MANUAL

3. Discussions of violation of biosafety protocols or procedures with the appropriate

persons.

4. Verification that all staff have received appropriate biosafety training.

5. Provision of continuing education in biosafety.

6. Investigation of incidents involving the possible escape of potentially infectious or

toxic material, and reporting of findings and recommendations to the laboratory

director and biosafety committee.

7. Coordination with medical staff regarding possible laboratory-acquired infections.

8. Ensuring appropriate decontamination following spills or other incidents involving

infectious material(s).

9. Ensuring proper waste management.

10. Ensuring appropriate decontamination of any apparatus prior to repair or servicing.

11. Maintaining awareness of community attitudes regarding health and environmental

considerations.

12. Establishment of appropriate procedures for import/export of pathogenic material

to/from the laboratory, according to national regulations.

13. Reviewing the biosafety aspects of all plans, protocols and operating procedures

for research work involving infectious agents prior to the implementation of these

activities.

14. Institution of a system to deal with emergencies.

Biosafety committee

A biosafety committee should be constituted to develop institutional biosafety policies

and codes of practice. The biosafety committee should also review research protocols

for work involving infectious agents, animal use, recombinant DNA and genetically

modified materials. Other functions of the committee may include risk assessments,

formulation of new safety policies and arbitration in disputes over safety matters.

The membership of the biosafety committee should reflect the diverse occupational

areas of the organization as well as its scientific expertise. The composition of a basic

biosafety committee may include:

1. Biosafety officer(s)

2. Scientists

3. Medical personnel

4. Veterinarian(s) (if work with animals is conducted)

5. Representatives of technical staff

6. Representatives of laboratory management.

The biosafety committee should seek advice from different departmental and specialist

safety officers (e.g. with expertise in radiation protection, industrial safety, fire

prevention, etc.) and may at times require assistance from independent experts in

various associated fields, local authorities and national regulatory bodies. Community

members may also be helpful if there is a particularly contentious or sensitive protocol

under discussion.

• 119 •

20. Safety for support staff

The safe and optimum operation of a laboratory is dependent to a great extent on the

support staff, and it is essential that such personnel are given appropriate safety training.

Engineering and building maintenance services

Skilled engineers and craftsmen who maintain and repair the structure, facilities and

equipment, should have some knowledge of the nature of the work of the laboratory,

and of safety regulations and procedures.

Testing of equipment after servicing, e.g. testing the efficiency of biological safety

cabinets after new filters have been fitted, may be carried out by or under supervision

of the biosafety officer.

Laboratories or institutions that do not have internal engineering and maintenance

services should establish good relationships with local service providers to familiarize

them with the equipment and work of the laboratory.

Engineering and maintenance staff should only enter Biosafety Level 3 or Biosafety

Level 4 laboratories with clearance and supervision by the biosafety officer and/or the

laboratory supervisor.

Cleaning (domestic) services

Biosafety Level 3 and Biosafety Level 4 laboratories should be cleaned by the laboratory

staff. Cleaning personnel should only enter Biosafety Level 3 or Biosafety Level 4

laboratories with clearance and supervision by the biosafety officer and/or the

laboratory supervisor.

21. Training programmes

A continuous, on-the-job safety training programme is essential to maintain safety

awareness among 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 organization’s goals and objectives.

The following are critical elements for an effective biosafety training programme.

1. 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.

2. Establishing training objectives. These are observable behaviours that the trainee

is expected to demonstrate, on the job, after the training. Objectives may acknow-

ledge the conditions under which certain activities or behaviours are performed

and the required level of proficiency.

3. 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.

4. 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. 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.

• 120 •

5. 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 in-

struction is to develop capabilities in problem-solving techniques, the instructional

approach should stress thinking/reasoning approaches rather than rote memori-

zation. The instruction provided should require productive behaviour and/or ap-

propriate 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.

6. 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 organization’s 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 may bear little relationship to the extent

of actual learning. It should not be used as the sole measurement of training

effectiveness.

7. 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.

WHO provides various tools for microbiological safety training.

21. TRAINING PROGRAMMES

• 121 •

PART VIII

Safety checklist

22. Safety checklist

• 125 •

This checklist is intended to assist in assessments of microbiological laboratory safety

and security status of biomedical laboratories.

Laboratory premises

1. Have guidelines for commissioning and certification been considered for facility

construction or post-construction evaluations?

2. Do the premises meet national and local building requirements, including those

relating to natural disaster precautions if necessary?

3. Are the premises generally uncluttered and free from obstructions?

4. Are the premises clean?

5. Are there any structural defects in floors?

6. Are floors and stairs uniform and slip-resistant?

7. Is the working space adequate for safe operation?

8. Are the circulation spaces and corridors adequate for the movement of people and

large equipment?

9. Are the benches, furniture and fittings in good condition?

10. Are bench surfaces resistant to solvents and corrosive chemicals?

11. Is there a hand-washing sink in each laboratory room?

12. Are the premises constructed and maintained to prevent entry and harbourage of

rodents and arthropods?

13. Are all exposed steam and hot water pipes insulated or guarded to protect personnel?

14. Is an independent power support unit provided in case of power breakdown?

15. Can access to laboratory areas be restricted to authorized personnel?

16. Has a risk assessment been performed to ensure that appropriate equipment and

facilities are available to support the work being considered?

Storage facilities

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?

3. Are freezers and storage areas lockable?

• 126 •

LABORATORY BIOSAFETY MANUAL

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?

4. Are there HEPA filters in the ventilation system?

5. Does mechanical ventilation compromise airflows in and around biological safety

cabinets and fume cupboards?

Lighting

1. Is the general illumination adequate (e.g. 300–400 lx)?

2. Is task (local) lighting provided at work benches?

3. Are all areas well-lit, with no dark or ill-lit corners in rooms and corridors?

4. Are fluorescent lights parallel to the benches?

5. Are fluorescent lights colour-balanced?

Services

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. Is the access of engineering and maintenance personnel to various laboratory areas

controlled and documented?

• 127 •

6. 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?

7. Are cleaning services available?

8. Is the access of cleaning personnel to various laboratory areas controlled and

documented?

9. Are information technology services available and secured?

Laboratory biosecurity

1. Has a qualitative risk assessment been performed to define risks that a security

system should protect against?

2. Have acceptable risks and incidence response planning parameters been defined?

3. Is the whole building securely locked when unoccupied?

4. Are doors and windows break-proof?

5. Are rooms containing hazardous materials and expensive equipment locked when

unoccupied?

6. Is access to such rooms, equipment and materials appropriately controlled and

documented?

Fire prevention and fire protection

1. Is there a fire alarm system?

2. Are the fire doors in good order?

3. Is the fire detection system in good working order and regularly tested?

4. Are fire alarm stations accessible?

5. Are all exits marked by proper, illuminated signs?

6. Is access to exits marked where the routes to them are not immediately visible?

7. Are all exits unobstructed by decorations, furniture and equipment, and unlocked

when the building is occupied?

8. Is access to exits arranged so that it is not necessary to pass through a high-hazard

area to escape?

9. Do all exits lead to an open space?

10. Are corridors, aisles and circulation areas clear and unobstructed for movement of

staff and fire-fighting equipment?

11. Is all fire-fighting equipment and apparatus easily identified by an appropriate

colour code?

12. Are portable fire extinguishers maintained fully charged and in working order,

and kept in designated places at all times?

13. Are laboratory rooms with potential fire hazards equipped with appropriate

extinguishers and/or fire blankets for emergency use?

14. If flammable liquids and gases are used in any room, is the mechanical ventilation

sufficient to remove vapours before they reach a hazardous concentration?

15. Are personnel trained to respond to fire emergencies?

22. SAFETY CHECKLIST

• 128 •

LABORATORY BIOSAFETY MANUAL

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?

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?

13. Are personnel trained to properly use and transport flammable liquids?

Compressed and liquefied gases

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?

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?

8. Are personnel trained to properly use and transport compressed and liquefied gases?

Electrical hazards

1. Are all new electrical installations and all replacements, modifications or repairs

made and maintained in accordance with a national 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)?

• 129 •

Personal protection

1. Is protective clothing of approved design and fabric 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 respirators available, regularly cleaned, disinfected, inspected and stored in a

clean and sanitary condition?

8. Are appropriate filters provided for the correct types of respirators, e.g. HEPA filters

for microorganisms, appropriate filters for gases or particulates?

9. Are respirators fit-tested?

Health and safety of staff

1. Is there an occupational health service?

2. Are first-aid boxes provided at strategic locations?

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?

13. Are personnel trained to follow appropriate biosafety practices?

14. Are laboratory staff encouraged to report potential exposures?

22. SAFETY CHECKLIST

• 130 •

LABORATORY BIOSAFETY MANUAL

Laboratory equipment

1. Is all equipment certified safe for use?

2. Are procedures available for decontaminating equipment prior to maintenance?

3. Are biological safety cabinets and fume cupboards regularly tested and serviced?

4. Are autoclaves and other pressure vessels regularly inspected?

5. Are centrifuge buckets and rotors regularly inspected?

6. Are HEPA filters regularly changed?

7. Are pipettes used instead of hypodermic needles?

8. Is cracked and chipped glassware always discarded and not reused?

9. Are there safe receptacles for broken glass?

10. Are plastics used instead of glass where feasible?

11. Are sharps disposal containers available and being used?

Infectious materials

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 and other protective clothing worn for unpacking specimens?

5. Are personnel trained to ship infectious substances according to current national

and/or international regulations?

6. Are work benches kept clean and tidy?

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 the appropriate chemical, physical and

biological indicators?

10. Is there a procedure for decontaminating centrifuges regularly?

11. Are sealed buckets provided for centrifuges?

12. Are appropriate disinfectants being used? Are they 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. Are incompatible chemicals effectively separated when stored or handled?

2. Are all chemicals correctly labelled with names and warnings?

3. Are chemical hazard warning charts prominently displayed?

4. Are spill kits provided?

5. Are staff trained to deal with spills?

6. Are flammable substances correctly and safely stored in minimal amounts in

approved cabinets?

• 131 •

7. Are bottle carriers provided?

8. Is a radiation protection officer or appropriate reference manual available for

consultation?

9. Are staff appropriately trained to safely work with radioactive materials?

10. Are proper records of stocks and use of radioactive substances maintained?

11. Are radioactivity screens provided?

12. Are personal radiation exposures monitored?

22. SAFETY CHECKLIST

PART IX

References, annexes

and index

References

• 135 •

1. Safety in health-care laboratories. Geneva, World Health Organization, 1997, (http://

whqlibdoc.who.int/hq/1997/WHO_LAB_97.1.pdf).

2. Garner JS, Hospital Infection Control Practices Advisory Committee. Guideline for isola-

tion precautions in hospitals. American Journal of Infection Control, 1996, 24:24–52, (http:/

/www.cdc.gov/ncidod/hip/isolat/isolat.htm).

3. Hunt GJ, Tabachnick WJ. Handling small arbovirus vectors safely during biosafety level 3

containment: Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) and exotic blue-

tongue viruses. Journal of Medical Entomology, 1996, 33:271–277.

4. National Research Council. Occupational health and safety in the care and use of research

animals. Washington, DC, National Academy Press, 1997.

5. 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.

6. Biosafety in microbiological and biomedical laboratories, 4th ed. Washington, DC, United

States Department of Health and Human Services/Centers for Disease Control and Pre-

vention/National Institutes of Health, 1999.

7. Class II (laminar flow) biohazard cabinetry. Ann Arbor, MI, National Sanitation Founda-

tion, 2002 (NSF/ANSI 49–2002).

8. Richmond JY, McKinney RW. Primary containment for biohazards: selection, installation

and use of biological safety cabinets, 2nd ed. Washington, DC, United States Department of

Health and Human Services/Centers for Disease Control and Prevention/National Insti-

tutes of Health, 2000.

9. Microbiological safety cabinets. Recommendations for information to be exchanged between

purchaser, vendor and installer and recommendations for installation. London, British Stand-

ards Institution, 1992 (Standard BS 5726–2:1992).

10. Microbiological safety cabinets. Recommendations for selection, use and maintenance. Lon-

don, British Standards Institution, 1992 (Standard BS 5726–4:1992).

11. Biological containment cabinets (Class I and II): installation and field testing. Toronto, Cana-

dian Standards Association, 1995 (Standard Z316.3–95 (R2000)).

12. Collins CH, Kennedy DA. Laboratory acquired infections: history, incidence, causes and pre-

vention, 4th ed. Oxford, Butterworth-Heinemann, 1999.

13. Health Canada. Laboratory biosafety manual, 2nd ed. Ottawa, Minister of Supply and Serv-

ices Canada, 1996.

14. Biological safety cabinets – biological safety cabinets (Class I) for personnel and environment

protection. Sydney, Standards Australia International, 1994 (Standard AS 2252.1–1994).

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ard AS 2252.2–1994).

• 136 •

LABORATORY BIOSAFETY MANUAL

16. Standards Australia/Standards New Zealand. Biological safety cabinets – installation and

use. Sydney, Standards Australia International, 2000 (Standard AS/NZS 2647:2000).

17. Advisory Committee on Dangerous Pathogens. Guidance on the use, testing and mainte-

nance of laboratory and animal flexible film isolators. London, Health and Safety Executive,

1990.

18. Standards Australia/Standards New Zealand. Safety in laboratories – microbiological aspects

and containment facilities. Sydney, Standards Australia International, 2002 (Standard AS/

NZS 2243.3:2002).

19. Centers for Disease Control and Prevention. Recommendations for prevention of HIV trans-

mission in health-care settings. Morbidity and Mortality Weekly Report, 1987, 36 (Suppl.

2):1S–18S.

20. Bosque PJ et al. Prions in skeletal muscle. Proceedings of the National Academy of Sciences of

the United States of America, 2002, 99:3812–3817.

21. Bartz JC, Kincaid AE, Bessen RA. Rapid prion neuroinvasion following tongue infection.

Journal of Virology, 2003, 77:583–591.

22. Thomzig A et al. Widespread PrPSc accumulation in muscles of hamsters orally infected

with scrapie. EMBO Reports, 2003, 4:530–533.

23. Glatzel M et al. Extraneural pathologic prion protein in sporadic Creutzfeld-Jakob disease.

New England Journal of Medicine, 2003, 349:1812–1820.

24. Brown P, Wolff A, Gajdusek DC. A simple and effective method for inactivating virus infec-

tivity in formalin-fixed tissue samples from patients with Creutzfield-Jakob disease. Neu-

rology, 1990, 40:887–890.

25. Taylor DM et al. The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed

brain-tissue. Veterinary Microbiology, 1997, 58:167–174.

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biomedical laboratories, 4th ed. Washington, DC, United States Department of Health and

Human Services, 1999:134–143.

27. Bellinger-Kawahara C et al. Purified scrapie prions resist inactivation by UV irradiation.

Journal of Virology, 1987, 61:159–166.

28. Health Services Advisory Committee. Safe working and the prevention of infection in clinical

laboratories. London, HSE Books, 1991.

29. Russell AD, Hugo WB, Ayliffe GAJ. Disinfection, preservation and sterilization, 3rd ed. Ox-

ford, Blackwell Scientific, 1999.

30. Ascenzi JM. Handbook of disinfectants and antiseptics. New York, NY, Marcel Dekker, 1996.

31. Block SS. Disinfection, sterilization & preservation, 5th ed. Philadelphia, PA, Lippincott

Williams & Wilkins, 2001.

32. Rutala WA. APIC guideline for selection and use of disinfectants. 1994, 1995, and 1996

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33. Sattar SA, Springthorpe VS, Rochon M. A product based on accelerated and stabilized hy-

drogen peroxide: evidence for broad-spectrum germicidal activity. Canadian Journal of

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Disinfection & sterilization in health care. Champlain, NY, Polyscience, 1997:79–92.

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in health care. Champlain, NY, Polyscience, 1997:273–280.

• 137 •

36. Steelman VM. Activity of sterilization processes and disinfectants against prions. In: Rutala

WA, e ds. Disinfection & sterilization in health care. Champlain, NY, Polyscience, 1997:255–

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37. Taylor DM. Transmissible degenerative encephalopathies: inactivation of the unconven-

tional causal agents. In: Russell AD, Hugo WB, Ayliffe GAJ, eds. Disinfection, preservation

and sterilization, 3rd ed. Oxford, Blackwell Scientific, 1999:222–236.

38. Infection control guidelines for hand washing, cleaning, disinfection and sterilization in health

care, 2nd ed. Ottawa, Laboratory Centre for Disease Control, Health Canada, 1998.

39. Springthorpe VS, Sattar SA. Chemical disinfection of virus-contaminated surfaces. CRC

Critical Reviews in Environmental Control, 1990, 20:169–229.

40. Recommendations on the transport of dangerous goods, 13th revised edition, New York and

Geneva, United Nations, 2003, (http://www.unece.org/trans/danger/publi/unrec/rev13/

13files_e.html).

41. Technical instructions for the safe transport of dangerous goods by air, 2003–2004 Edition.

Montreal, International Civil Aviation Organization, 2002.

42. Economic Commission for Europe Inland Transport Committee. Restructured ADR appli-

cable as from 1 January 2003. New York and Geneva, United Nations, 2002, (http://

www.unece.org/trans/danger/publi/adr/adr2003/ContentsE.html).

43. Infectious substances shipping guidelines. Montreal, International Air Transport Association,

2003, (http://www.iata.org/ads/issg.htm).

44. Transport of Infectious Substances. Geneva, World Health Organization, 2004, (http://

www.who.int/csr/resources/publications/WHO_CDS_CSR_LYO_2004_9/en/).

45. Berg P et al. Asilomar conference on recombinant DNA molecules. Science, 1975, 188:991–

994.

46. 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 Jour-

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47. O’Malley BW Jr et al. Limitations of adenovirus-mediated interleukin-2 gene therapy for

oral cancer. Laryngoscope, 1999, 109:389–395.

48. World Health Organization. Maintenance and distribution of transgenic mice susceptible

to human viruses: memorandum from a WHO meeting. Bulletin of the World Health Or-

ganization, 1993, 71:497–502.

49. Furr AK. CRC handbook of laboratory safety, 5th ed. Boca Raton, FL, CRC Press, 2000.

50. Lenga RE. The Sigma-Aldrich Library of Chemical Safety Data, 2nd ed. Milwaukee, WI,

Aldrich Chemical Company, 1988.

51. Lewis RJ. Sax’s dangerous properties of industrial materials, 10th ed. Toronto, John Wiley

and Sons, 1999.

REFERENCES

ANNEX 1

First aid

First aid is the skilled application of accepted principles of medical treatment at the

time and place of an accident. It is the approved method of treating a casualty until he

or she is placed in the care of a doctor for definitive treatment of the injury.

The minimum first-aid equipment consists of a first-aid box, protective clothing

and safety equipment for the person rendering the first aid, and eye irrigation

equipment.

The first-aid box

The first-aid box should be constructed from materials that will keep the contents

dust- and damp-free. It should be kept in a prominent position and be easily recognized.

By international convention, the first-aid box is identified by a white cross on a green

background.

The first-aid box should contain:

1. Instruction sheet giving general guidance

2. Individually-wrapped sterile adhesive dressings in a variety of sizes

3. Sterile eye-pads with attachment bandages

4. Triangular bandages

5. Sterile wound coverings

6. Safety pins

7. A selection of sterile but unmedicated wound dressings

8. An authoritative first-aid manual, e.g. one issued by the International Red Cross.

Protective equipment for the person rendering first aid includes:

1. Mouthpiece for mouth-to-mouth resuscitation

2. Gloves and other barrier protections against blood exposures,1 and

3. Clean-up kit for blood spills (see Chapter 14 of the manual).

Eye irrigation equipment should also be readily available and staff trained in its correct

use.

1 Garner JS, Hospital Infection Control Practices Advisory Committee. Guideline for isolation precautions

in hospitals. American Journal of Infection Control, 1996, 24:24–52, (http://www.cdc.gov/ncidod/hip/

isolat/isolat.htm).

• 138 •

ANNEX 2

Immunization of staff

The risks of working with particular agents should be fully discussed with individual

researchers. The local availability, licensing state and utility of possible vaccines and/

or therapeutic drugs (e.g. antibiotic treatments) in case of exposure should be evaluated

before work with such agents is started. Some workers may have acquired immunity

from prior vaccination or infection.

If a particular vaccine or toxoid is locally licensed and available, it should be offered

after a risk assessment of possible exposure and a clinical health assessment of the

individual have been carried out.

Facilities for specific clinical case management following accidental infections should

also be available.

• 139 •

ANNEX 3

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, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzer-

land (http://www.who.int/csr/).

•WHO Collaborating Centre for Biological Safety, Swedish Institute for Infectious

Disease Control, Nobels Väg 18, S-171 82 Solna, Sweden

(http://www.smittskyddsinstitutet.se/English/english.htm).

•WHO Collaborating Centre on Biosafety Technology and Consultative Services,

Office of Laboratory Security, Health Canada, 100 Colonnade Road, Loc.: 6201A,

Ottawa, Ontario, Canada K1A 0K9 (http://www.hc-sc.gc.ca/pphb-dgspsp/ols-bsl).

•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,

Division of Occupational Health and Safety, Office of Research Services, National

Institutes of Health, Department of Health and Human Services, 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 ad-

dress: Locked Bag 815, PO Carlton Sth, Victoria 3053, Australia (http://www.

vidrl.org.au/).

• 140 •

ANNEX 4

Equipment safety

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 11 of the manual).

Equipment that may create a hazard

Table A4-1 lists equipment and operations that may create hazards and suggests how

such hazards may be eliminated or reduced.

Table A4-1. Equipment and operations that may create hazards

EQUIPMENT HAZARD HOW TO ELIMINATE OR REDUCE THE HAZARD

Hypodermic Accidental inoculation, • Do not recap or clip needles.

needles aerosol or spillage • 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.

•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.

•Use a biological safety cabinet for all

operations with infectious material.

• 141 •

• 142 •

LABORATORY BIOSAFETY MANUAL

EQUIPMENT HAZARD HOW TO ELIMINATE OR REDUCE THE HAZARD

•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 • Use sealable buckets (safety cups) or sealed

and tube breakage rotors. Open buckets or rotors after aerosols

have settled (30 min) or in a biological safety

cabinet.

Ultra-centrifuges Aerosols, splashing • Install a HEPA filter between centrifuge and

and tube breakage vacuum 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.

Anaerobic jars Explosion, dispersing • Ensure integrity of wire capsule around

infectious materials catalyst.

Desiccators Implosion, dispersing • Place in a stout wire cage.

glass fragments and

infectious materials

Homogenizer, Aerosols, leakage and • Operate and open equipment in a biological

tissue grinders container breakage 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.

Sonicators, Aerosols, impaired • Operate and open equipment in a biological

ultrasonic hearing, dermatitis safety cabinet or sealed unit.

cleaners • Ensure insulation to protect against

subharmonics.

•Wear gloves to protect skin against chemical

effects of detergents.

• 143 •

EQUIPMENT HAZARD HOW TO ELIMINATE OR REDUCE THE HAZARD

Culture stirrers, Aerosols, splashing • Operate in a biological safety cabinet or

shakers, agitators and spillage specially designed primary containment.

•Use heavy-duty screw-capped culture flasks,

fitted with filter-protected outlets, if

necessary, and well secured.

Freeze-dryers Aerosols and direct • Use O-ring connectors to seal the unit

(lyophilizers) contact contamination 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.

Water baths Growth of micro- • Ensure regular cleaning and disinfection.

organisms. Sodium • Do not use sodium azide for preventing

azide forms explosive growth of organisms.

compounds with

some metals.

In addition to microbiological hazards, safety hazards associated with equipment

should also be anticipated and prevented. Table A4-2 lists examples of some of the

causes of accidents.

ANNEX 4. EQUIPMENT SAFETY

• 144 •

LABORATORY BIOSAFETY MANUAL

Table A4-2. Common causes of equipment-related accidents

ACCIDENT ACCIDENT CAUSE REDUCING OR ELIMINATING THE HAZARD

Faulty design or construction

Electrical fires in No over-temperature cut-out • Compliance with national

incubators standards.

Electrical shock Failure to provide reliable

earthing/grounding

Improper use

Centrifuge accident Failure to balance buckets • Train and supervise staff.

on swing-out rotors

Anaerobic incubator Use of incorrect gas • Train and supervise staff.

explosion

Improper adaptation

Explosion in domestic Improper transport of liquid • Use of specially designed

vacuum flask nitrogen equipment.

Explosion in domestic- Dangerous chemical not • Store low-flashpoint solvents

type refrigerator stored in spark-/explosion- and extracts only in spark-/

proof container, explosion-proof refrigerators or

e.g. diethyl ether with cabinets.

leaking screw cap

Lack of proper maintenance

Fire in flame Incorrect reassembly of • Train and supervise staff.

photometer components during

maintenance

• 145 •

ANNEX 5

Chemicals: hazards

and precautions

This annex lists the basic health and safety information, data and appropriate safety

precautions for a selected number of chemicals found commonly in health-care and

research laboratories. The list is not exhaustive and the absence of any particular

chemical does not imply that it is non-hazardous. All laboratory chemicals should be

treated with caution and in ways that will minimize exposure.

• 146 •

LABORATORY BIOSAFETY MANUAL

Table A5-1. Chemicals: hazards and precautions

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Acetaldehyde Colourless liquid or Mild eye and Extremely flammable; No open flames, no Can form explosive

CHO gas with a pungent, respiratory tract vapour/air mixtures sparks, no smoking, peroxides in contact

fruity odour; irritation. Effects on are explosive; no contact with hot with air. May polymerize

m.p. –121 °Cthe central nervous flash point –39 °Csurfaces. Store in under influence of acids,

b.p. 21 °C. system, respiratory flammable range tightly sealed alkaline materials, in

tract and kidneys. 4–57%. containers in areas the presence of trace

Possible carcinogen. separate from metals. A strong

oxidizers; store only if reducing agent, reacts

stabilized. Use in violently with oxidants,

exhaust cupboard or with various organic

with good ventilation. substances, halogens,

Wear rubber gloves, sulfuric acid and

safety goggles, and amines.

respiratory protection.

Acetic acid Colourless liquid with Corrosive; causes Flammable; Do not breathe fumes. Violent or explosive

Hpungent odour; severe burns; irritating flashpoint 40 °CIn case of contact with reaction with oxidizers.

m.p. 17 °Cvapour. Effects may flammable range eyes rinse immediately

b.p. 118 °C; be delayed. 5.4–16%. with water and seek

miscible with water.medical advice. Wear

nitrile gloves and eye

protection.

Acetic Colourless liquid with Severe irritation of Flammable; evolves No open flames, no Reacts violently with

anhydride a strong pungent, eyes and upper irritation or toxic sparks, no smoking. boiling water, steam,

(CH

CO)

Ovinegar-like odour; respiratory tract fumes or gases in a Prevent skin and eye strong oxidants, alcohols,

m.p. –73 °Cirritation; corrosive fire; contact. amines, strong bases and

b.p. 139 °C. action. Effects may be flashpoint 49 °Cmany other compounds.

delayed. explosive limits Attacks many metals

2.7–10.3%. in presence of water.

• 147 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Acetone Colourless volatile Slight eye, nose and Highly flammable; Keep container in Reacts violently with Earth/ground large

COCH

liquid with sweetish throat irritation. flashpoint –18 °Cwell-ventilated area; oxidizers (e.g. chromic containers and

odour; Inhalation may cause explosive limits keep away from and nitric acids) and vessels to prevent

m.p. –95 °C, dizziness, narcosis 2.2–12.8%. sources of ignition. chloroform in the static electricity.

b.p. 56 °C.; and coma. Do not breathe vapour. presence of base.

miscible with water.Use respiratory Incompatible with

protection; wear eye concentrated sulfuric

protection. and nitric acid mixtures.

Acetonitrile Colourless liquid with Respiratory, eye and Highly flammable; No open flames, no Reacts with aqueous

CN an aromatic odour; skin irritation. flashpoint 12.8 °Csparks, no smoking, acids and bases,

m.p –46 °CExposure may result in explosive limits no contact with oxidants. producing toxic

b.p. 82 °C. convulsions 3.0–16%. Use only in areas free of fumes. Reacts with

unconsciousness, ignition sources. Store strong oxidants.

cyanide poisoning. in tightly sealed Attacks some forms

containers in areas of plastic, rubber and

separate from oxidizers. coatings. Decomposes

Work with exhaust on burning producing

ventilation. Avoid skin, hydrogen cyanide and

eye and mucous nitrogen oxides.

membrane contact. Use

respiratory protection and

rubber gloves.

Acetylene Colourless gas with Simple asphyxiant; Extremely flammable; For skin protection use Strong reducing agent;

HC≡CH a faint, ethereal or frostbite on skin flammable range cold-insulating gloves reacts violently with

garlic-like odour; contact. 2.5–100%. and safety goggles or oxidants and with

shipped under pressure, face shield. No open fluorine or chlorine

dissolved in acetone; flames, no sparks, no under influence of light.

m.p. –81 °Csmoking. Work with Reacts with copper,

sublimes at –84 °C. local exhaust ventilation,silver and mercury or

explosion-proof electrical their salts, forming shock-

equipment and lighting. sensitive compounds.

• 148 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Acrolein Colourless or yellow Lacrimation. Severe Highly flammable; Prevent skin and eye Oxidizers, acids, alkalis,

=CHCHO liquid with a piercing, respiratory irritation; flashpoint –26 °Ccontact. Work in fume ammonia, amines. Poly-

disagreeable odour; lung oedema at high explosive limits cupboard or with good merizes readily unless

m.p. –87 °Cexposure levels. 2.8–31%. ventilation. inhibited, usually with

b.p. 53 °C. Effects may be delayed. hydroquinone. May

form shock-sensitive

peroxides over time.

Ammonia Colourless liquid with Corrosive to eyes, As ammonia gas; Keep container tightly Reacts violently with

solutions pungent odour; for gas: respiratory system flammable range closed. In case of contact heavy metals such as

m.p. –78 °Cand skin on ingestion; 15–28%. with eyes, rinse mercury and their salts

b.p. –33 °C; lung oedema at high immediately and seek to form explosive

for 25% solution: levels of exposure to medical advice. Work in products.

m.p. –58 °Cgas or vapour. fume cupboard. Wear

b.p. 38 °C; rubber or plastic gloves and

miscible with water.chemical-grade goggles.

Aniline Colourless to brown, Cyanosis due to Combustible; Store in tightly sealed Strong oxidizers,

oily liquid with an methaemoglobinaemia. flashpoint 70 °Ccontainers in areas strong acids.

aromatic amine-like Eye and skin irritation. explosive range separate from oxidizers.

odour; May be absorbed 1.2–11%. Prevent skin and eye

m.p. –6 °Cthrough the skin; contact. Work with local

b.p. 185 °C. repeated or prolonged exhaust ventilation or

exposure may cause respiratory protection,

sensitization. protective gloves, protective

clothing, face shield.

Auramine Yellow flakes or Harmful by ingestion, Avoid skin contact and Strong oxidizing agents.

4,4'-Carbono- powder; inhalation and skin inhalation of dust. Wear

imidoylbis m.p. 136 °C; contact. May cause rubber or plastic gloves

(N,N-dimethyl- insoluble in water. eye or skin irritation. and chemical-grade goggles.

benzenamine) Possible carcinogen. Work in fume cupboard or

wear dust respirator.

• 149 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Benzene Colourless volatile Inhalation of vapour Highly flammable; Keep container in well- Can react violently

liquid with causes effects on flashpoint –11 °Cventilated area and away with oxidizers

characteristic central nervous flammable range from sources of ignition. including chromic acid,

aromatic odour; system resulting in 1.3–8%. Work in fume cupboardpotassium

m.p. 6 °Cvertigo and headache; or hood with adequate permanganate and

b.p. 80 °C. at high concentrations, ventilation. Wear eye liquid oxygen.

unconsciousness and protection and nitrile or

death. Risk of aplastic PVC gloves. Prevent

anaemia, leukaemia, formation of electrical

liver damage on charges by earthing/

prolonged or chronic grounding.

exposure. May be

absorbed through skin.

Benzidine Light yellow powder; May be absorbed Combustible, gives Avoid all exposure. Use is prohibited or

1,1'-Biphenyl- m.p. 128 °Cthrough skin. May off toxic fumes Wear eye and skin legally controlled in

4,4'-diamine b.p. 400 °Ccause bladder cancer.(gases) in a fire. protection. Work in fume many countries.

slightly soluble in Avoid all exposure. cupboard with exhaust

water but very soluble ventilation.

in acids and organic

solvents.

Bromine Dark reddish-brown Corrosive. Vapour is Not combustible but Use in closed system Strong oxidant, Attacks some

fuming liquid with corrosive to eyes and enhances combustion and with ventilation. reacts violently with forms of plastic,

pungent odour; respiratory tract; of other substances. Wear protective gloves combustible and rubber and

m.p. –7.2 °Cinhalation may cause Many reactions may and clothing, safety reducing materials. coatings.

b.p. 58.8 °C. lung oedema and cause fire or goggles, face shield Reacts violently with

effects on central explosion. Heating or eye protection in aqueous ammonia,

nervous system. Eye will cause rise in combination with oxidants, metals,

contact can cause pressure with risk respiratory protection. organic compounds

blurred vision, redness, of burning. and phosphorus.

pain, severe tissue

burns.

• 150 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Carbon dioxide Translucent white Risk of asphyxiation Wear protective Alkali metals, strong

(solid; “dry solid at –79 °C; in confined or poorly insulated gloves. Store bases.

ice”) CO

sublimes to gas at ventilated areas; contact only in ventilated room

ambient temperature. with solid “dry ice” or area in open

causes frostbite. container.

Carbon tetra- Colourless liquid with May be absorbed Not combustible. Avoid all contact. Work On contact with hot

chloride characteristic ether-through skin; may Gives off irritating with ventilation, local surfaces or flames,

CCl

like odour; cause dermatitis on or toxic fumes or exhaust or respiratory decomposes forming

m.p. –23 °Cprolonged exposure. gases in a fire. protection; use nitrile toxic and corrosive

b.p. 76.5 °C. Eye irritation. May gloves and protective fumes and gases

cause liver and kidney clothing, face shield or (hydrogen chloride,

damage and central eye protection in chlorine, phosgene).

nervous system combination with Reacts with some

disturbances resulting respiratory protection. metals such as

in headache, nausea, aluminium,

slight jaundice, loss of magnesium, zinc.

appetite and narcosis.

An animal carcinogen.

Chlorine Greenish-yellow gas Corrosive to eyes, skin Not combustible but Work with closed system Solution in water is a Attacks many

with pungent odour; and respiratory tract. enhances and ventilation. Wear strong acid, reacts metals in

m.p. –101 °CInhalation of gas may combustion of other cold-insulating gloves, violently with bases presence of water.

b.p. –34 °C. cause pneumonitis and substances. protective clothing, and many organic Attacks plastics,

lung oedema, resulting safety goggles or eye compounds, acetylene, rubber and

in reactive airways dys- protection in combination butadiene, benzene coatings.

function syndrome (RADS). with respiratory and other petroleum

Rapid evaporation of the protection. fractions, ammonia,

liquid may cause frostbite. hydrogen, sodium

High exposures may carbide, turpentine

result in death. Effects and finely divided

may be delayed; medical metals causing fire

observation indicated. and explosion hazard.

• 151 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Chlorine Yellow to red gas or Severe irritation of Not combustible Work in closed system A strong oxidant; reacts

dioxide ClO

a red-brown liquid; eyes, skin and but enhances with ventilation. Wear violently with combustible

m.p –59 °Crespiratory tract; combustion of other protective gloves and and reducing materials.

b.p. 10 °C. inhalation of gas may substances; may clothing, safety goggles Reacts violently with

cause lung oedema. explode on heating, or eye protection in phosphorus, potassium

Effects may be delayed; on exposure to sun- combination with hydroxide, sulfur,

medical observation light or if subjected respiratory protection. ammonia, methane,

indicated. to shock and sparks. phosphine and hydrogen

sulfide.

Chloroform Colourless volatile Harmful by inhalation, Wear protective Strong bases; some When heated to

CHCl

liquid with ingestion and skin contact; clothing, nitrile gloves metals such as decomposition,

characteristic odour; skin irritation. May cause and eye protection. aluminium or forms phosgene

m.p. –63 °Ceffects on liver, kidneys Work in a fume magnesium, zinc gas.

b.p. 61 °C; and central nervous cupboard. powder; strong Attacks plastics,

slightly soluble in system resulting in oxidizers. rubber.

water. headache, nausea, slight

jaundice, loss of appetite,

narcosis. Prolonged or

chronic exposure causes

cancer in animals; sus-

pected human carcinogen.

Chromic acid Dark red odourless Irritation of eyes, skin Decomposes above Prevent skin and eye In aqueous solution is

CrO

flakes or powder often and respiratory 250 °C to chromic contact; avoid inhalation a strong acid which

Chromium VI used in aqueous system. Repeated or oxide and oxygen of fine dust and mist. reacts with bases and

oxide solutions; prolonged contact with with increased fire Work with ventilation, is corrosive. Strong

m.p. 197 °C. skin may cause derma- hazard. Many local exhaust or oxidant, reacts with

titis, chrome ulcers and reactions may cause respiratory protection. combustible, organic

skin sensitization. hazards. or other readily

Inhalation may cause oxidizable materials

asthma-like reactions. (paper, wood, sulfur,

May cause perforation of aluminium, plastics

nasal septum. Human etc.). Corrosive to

carcinogen. metals.

• 152 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Copper Reddish, lustrous, Inhalation of copper Combustible. Work with local exhaust Shock-sensitive

Cu malleable, odourless fume may cause metal or repiratory protection, compounds are formed

solid; red powder, fume fever. protective gloves and with acetylenic

turns green on goggles. compounds, ethylene

exposure to moist air; oxides azides and

m.p. 1083 °Chydrogen peroxide.

b.p. 2567 °C. Reacts with strong

oxidants like chlorates,

bromates and iodates,

causing explosion hazard.

Cyanogen Colourless or white Severe eye, skin and Not combustible but Work in closed system Decomposes on heating

bromide crystals, with respiratory tract forms flammable gas with ventilation. Wear and on contact with

BrCN pungent odour; effects; inhalation of on heating. Gives off protective gloves and acids producing highly

m.p. 52 °Cvapour may cause lung irritating or toxic protective clothing, toxic and flammable

b.p. 61 °C. oedema which may fumes or gases in safety goggles face hydrogen cyanide and

result in convulsions, a fire. shield or eye protection corrosive hydrogen

unconsciousness, in combination with bromide. Reacts with

respiratory failure and respiratory protection. strong oxidants. Reacts

death. slowly with water and

moisture to produce

hydrogen bromide and

hydrogen cyanide.

Attacks many metals in

the presence of water.

Cytochalasin White powder; Toxic by ingestion, Avoid contact with eyes, Strong oxidizing agents.

(A–J) m.p. varies. inhalation or absorption skin, clothing; wear

through skin. May chemical-grade goggles

cause congenital fetal and rubber or plastic

malformation. gloves.

• 153 •

Diethyl ether Colourless highly Irritation of eyes and Extremely flammable; Keep container in well- Exposure to air and

volatile liquid with respiratory tract . May flashpoint –45 °Cventilated area; keep light may result in

sweet characteristic affect central nervous flammable range away from sources of formation of explosive

odour; system causing 1.7–48%. ignition; earth/ground peroxides. Can react

m.p. –116 °Cdrowsiness and containers to prevent violently with oxidizers

b.p. 34 °C; unconsciousness. static electrical and halogens.

slightly soluble in Repeated inhalation discharges. Work in

water. may cause addiction. fume cupboard. Wear

nitrile gloves to prevent

defatting of skin.

Dimethylamine Colourless volatile Severe irritation of Extremely flammable; Keep away from sources Can react with oxidizers,

(CH

)

NH liquefied gas with eyes and respiratory flashpoint –26 °Cof ignition; in case of mercury.

pungent odour; system; inhalation flammable limits contact with eyes rinse

m.p. –93 °Cmay cause lung oedema. 2.8–14%. immediately and seek

b.p. 7 °C; Rapid evaporation may Solution highly medical advice. Work in

miscible with water.cause frostbite. flammable; fume cupboard. Wear

Solution is corrosive to flashpoint –18 °C. nitrile gloves and

eyes and skin. chemical-grade goggles.

2,4-Dinitro- Orange-red crystalline Irritation of skin and Keep moist to reduce Can react vigorously

phenyl-hydrazine powder; eyes. Harmful by explosion risk. Wear with oxidizers and

(NO

)

-m.p. 200 °C; ingestion, inhalation dust respirator, rubber reducers.

NHNH

slightly soluble in water.and skin contact. or plastic gloves and

1-Hydrazino- chemical-grade goggles.

2,4-dinitro-

benzene

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 154 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Dioxane Colourless liquid, Irritation of eyes and Highly flammable; Work with ventilation, Can form explosive

with characteristic respiratory tract. May distant ignition local exhaust. No open peroxides. Reacts

Diethylene odour; affect central nervous possible; as a result flames, no sparks, no vigorously with strong

dioxide m.p. 12 °Csystem resulting in of flow, agitation, smoking, no contact oxidants and

b.p. 101 °C. headache, nausea, etc., electrostatic with strong oxidants or concentrated strong

cough, sore throat, charges can be hot surfaces. Do not use acids. Reacts

abdominal pain, generated. compressed air for filling, explosively with some

dizziness, drowsiness, discharging or handling; catalysts. Attacks many

vomiting, unconscious- use non-sparking tools. plastics.

ness. May be absorbed Wear protective gloves,

through skin. Kidney clothing, face shield or

and liver damage. eye protection, in

Probably a human combination with

carcinogen. respiratory protection.

Ethanol Colourless volatile Harmful if ingested. Highly flammable; Keep container tightly Reacts violently with

OH liquid with slight, Irritation of eyes. May flashpoint 12 °Cclosed; keep away from strong oxidizers.

characteristic odour; affect central nervous flammable limits ignition sources.

m.p. –117 °Csystem. 3–19%.

b.p. 79 °C;

miscible with water.

Ethanolamine Colourless non-volatile Corrosive to eyes, Flashpoint 85 °C. Wear rubber or plastic Reacts with strong

NCH

OH viscous liquid with respiratory system gloves and eye oxidizers.

2-Amino- ammoniacal odour; and skin. May cause protection.

ethanol m.p 10 °Cskin sensitization.

b.p. 171 °C;

miscible with water.

• 155 •

Formaldehyde Colourless liquid with Severely irritation of Flashpoint 50 °C. Wear protective clothing Can react vigorously Concentrated

solution a pungent odour; eyes and skin, irritation such as plastic apron, with oxidizers, with formaldehyde

(37–41% b.p. 96 °C; of respiratory tract; pro- rubber or plastic gloves nitromethane to solutions become

formaldehyde miscible with water.longed exposure to the and chemical-grade produce explosive cloudy if stored

with 11–14% vapour may cause goggles. Work in fume products, with below 21 °C and

methanol) asthma-like symptoms, cupboard or well- hydrochloric acid to should be kept at

HCHO conjunctivitis, laryngitis, ventilated area. produce the potent 21–25 °C. Dilute

bronchitis or broncho- carcinogen

bis

solutions (1–5%)

pneumonia. May cause (chloromethyl) ether. and medium-

sensitization by skin strength solutions

contact. Possible risk (5–25%) retain

of irreversible health many of the

effects. Possible hazards of the

carcinogen. concentrated form.

Glutaraldehyde Colourless or pale Severe irritation of Work in fume cupboardCan react vigorously Often supplied in

OHC(CH

)

CHO yellow solution with eyes and upper or well-ventilated area. with oxidizers. aqueous solution

pungent odour; respiratory tract; Wear rubber or plastic at various concen-

m.p. –14 °Cprolonged inhalation gloves and eye trations with added

b.p. 189 °C; exposure or skin contact protection. stabilizer to

miscible with water.may cause sensitization. enhance stability.

Hydrochloric Colourless fuming Corrosive to eyes, Do not breathe fumes; Reacts violently with Releases highly

acid (10–37%) liquid with a pungent respiratory system use respiratory protection. bases (solids and toxic fumes in

HCl odour; and skin; repeated In case of contact with concentrated solutions), fires.

Hydrogen b.p. –121 °C; inhalation of vapour eyes, rinse immediately explosively with

chloride miscible with water.can cause chronic with water and seek solid potassium

bronchitis. medical advice; in case of permanganate. Gives

contact with skin, wash off toxic or explosive

immediately with plenty gases on contact with

of water. Work in fume many metals.

cupboard. Wear rubber

or plastic gloves and eye

protection (spectacles or

goggles).

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 156 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Hydrogen Colourless liquid; Corrosive at high Oxidizing agent; In case of contact with Reacts vigorously with Can decompose

peroxide m.p. –39 °C (70%) concentration (60%), contact with skin, wash immediately a variety of chemical evolving oxygen,

b.p. 125 °C (70%); and at low combustible material with plenty of water.reagents including causing pressure

miscible with water,concentration (6%) can cause fire. Wear nitrile gloves and oxidizers and bases. rise in container.

supplied in aqueous if contact with skin is eye protection if Attacks most metals Store in dark, cool

solution at various prolonged. Dilute concentration or their salts, flammable place. Do not use

concentrations. solutions are irritating exceeds 20%. liquids and other metallic containers

to eyes, respiratory combustible materials or equipment, e.g.

system and skin. (paper, textiles), aniline brass, copper,

and nitromethane. iron.

Hydrogen Colourless gas with May cause effects on Extremely flammable; Work with ventilation, Strong oxidizers and Sense of smell

sulfide a strong odour of central nervous system explosive limits local exhaust. Wear strong nitric acid. becomes rapidly

Srotten eggs; resulting in headache, 4.3–46%. safety goggles or eye Attacks many metals fatigued and

b.p. –60 °Cdizziness, cough, sore protection in and plastics. cannot be relied

m.p. –85 °C. throat, nausea, laboured combination with on to warn of the

breathing, unconscious- respiratory protection. continuous

ness and death. Inhala- presence of the

tion may cause lung gas.

oedema. Redness, pain,

severe deep burns of eyes.

Iodine Bluish-black Irritation of eyes, Not combustible Do not breathe vapour; Reacts violently with

crystalline scales with respiratory system but enhances avoid contact with metals including

a characteristic odour; and skin. Repeated combustion of other eyes. Wear nitrile aluminium, potassium

m.p. 114 °Cexposure may cause substances. Many gloves. and sodium, and with

b.p. 184 °C; skin sensitization. reactions may cause ethanol/phosphorus

practically insoluble May have effect on fire or explosion. Gives mixtures, acetylene

in water. thyroid. off irritating or toxic and ammonia.

fumes (or gases) in a fire.

• 157 •

Mercury Heavy silvery liquid; May be absorbed Not combustible. Keep container tightly Acetylene, fulminic Store containers

Hg m.p. –39 °Cthrough skin. Gives off irritating or closed. Work in fume acid. Reacts with and use over catch-

(Quicksilver) b.p. 357 °C; Repeated exposure toxic fumes in a fire. cupboard or well- ammonia, azides and ment trays to

insoluble in water.may affect kidneys ventilated area. Prevent ethylene oxide to form contain spillage;

and central nervous spills. Observe strict explosive products. suck up spilt drop-

system, and may cause hygiene. Wear nitrile Reacts violently with lets into a small

vomiting, diarrhoea, gloves. bromine. Forms respiratory bottle

headache, nausea, amalgams with many fitted with a capillary

swollen gums, loose metals. connecting tube

teeth. and connected to

a pump; treat spilt

areas with zinc dust

to form an amalgam.

Methanol Colourless volatile Effects on central Highly flammable; Keep container tightly Can react vigorously

OH liquid with nervous system result- flashpoint –16 °Cclosed; keep away from with oxidizers. Reactions

characteristic odour; ing in unconsciousness; flammable range ignition sources. Avoid with magnesium or

m.p. –98 °Cmucous membrane 7–37%. breathing vapour and bromine can be violent

b.p. 65 °C; irritation. Chronic contact with skin. Work and those with strong

miscible with water.exposure can cause in fume cupboard or well- oxidants or chloroform

damage to retina and ventilated area. Wear with sodium can be

optic nerve. Prolonged rubber or plastic gloves explosive.

skin contact may cause and eye protection.

dermatitis. May be

absorbed through skin.

Naphthylamine White to pink crystals Both forms very toxic Combustible. Avoid all exposure; wear Use is prohibited or

(alpha and beta) with characteristic odour; by inhalation, ingestion suitable protective legally controlled in

Nalpha: m.p. 50 °Cand skin contact. clothing. Work in fume many countries.

-phenyl-α-b.p. 301 °C; Human carcinogen cupboard or hood, or

naphthylamine beta: m.p. 113 °Ccausing bladder cancer.with exhaust ventilation.

and b.p. 306 °C; Experimental mutagen

-phenyl-β-poorly soluble in water and teratogen.

naphthylamine but hydrochloride is Absorbed through skin.

water-soluble.

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 158 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Ninhydrin Pale yellow solid, Harmful by ingestion Flammable, Avoid inhalation of the Contact with skin

decomposes before and inhalation. Irritation combustible solid; spray or vapour and produces a

melting at 241 °C. of eyes, respiratory flashpoint 39 °C. contact with eyes. Wear persistent violet

Supplied in aerosol system and skin. rubber or plastic gloves stain.

spray cans as 0.5% Repeated exposure may and chemical-grade

solution in butanol; cause skin sensitization. goggles.

soluble in water.

Nitric acid Colourless or pale Corrosive; causes Oxidizer; contact Do not breathe vapour; Acetic acid, chromic Concentrated nitric

(50–70%) yellow fuming liquid; severe burns to eyes with combustible use respiratory protection. acid, hydrocyanic acid, acid is involved in

HNO

m.p. –42 °Cand skin. Inhalation of material may cause In case of contact with aniline, carbon, more dangerous

b.p. 83–121 °C; vapour may cause lung fire. Evolves toxic eyes, rinse immediately hydrogen sulfide, bases, reactions than any

miscible in water. oedema. fumes in a fire. and seek medical attention; metals and many other other chemical

in case of contact with substances. reagent.

skin, wash off immediately;

remove contaminated

clothing. Wear PVC gloves,

plastic apron and chemical-

grade goggles. Work in

fume cupboard.

Nitrobenzene Pale yellow oily liquid, Methaemoglobinanaemia Combustible; risk of Work with ventilation, On combustion forms

with characteristic odour; with cyanosis, liver fire and explosion; local exhaust or corrosive fumes including

m.p. 6 °Cdamage; symptoms flashpoint 88 °C. respiratory protection. nitrogen oxides. Reacts

b.p. 211 °C. include blue lips or Wear protective violently with strong

fingernails, blue skin, gloves, protective oxidants and reducing

dizziness, nausea, clothing, safety goggles. agents, causing fire and

weakness, unconscious- explosion hazard. Attacks

ness. Absorbed through many plastics. Forms ex-

skin. plosive (thermally unstable)

substances or mixtures

with many organic and

inorganic compounds.

• 159 •

Osmium Pale yellow crystals with Very toxic by inhalation, Powerful oxidizing Keep container tightly

tetroxide pungent odour; ingestion and skin agent. Not combustible closed and in a well-

OsO

m.p. 40 °Ccontact, causing severe but enhances ventilated area. Work with

b.p. 130 °C; burns and irritation. combustion of other solid and solutions in fume

sublimes below boiling Vapour, solid and substances. cupboard or hood. Wear

point; soluble in water.solutions are corrosive chemical-grade goggles

to skin and respiratory and protective gloves. To

tract. Inhalation may make up solutions, add

cause lung oedema. unopened ampoule to

required volume of water,

stopper and shake to break

ampoule.

Oxalic acid Colourless crystals; Harmful if in contact Combustible. Gives Avoid contact with skin Oxidizing agents; also

CCO

Hsoluble in water; with skin or if ingested. off irritating or toxic and eyes; wear eye silver and mercury

m.p. 190 °C, Dust irritates respiratory fumes (or gases) in a protection and gloves. and their compounds.

decomposes. tract and eyes. Solutions fire.

irritate eyes and may

cause skin burns.

Oxygen Colourless compressed At very high concen- Not combustible but No open flames, no A strong oxidant, reacts

gas; trations, irritation of enhances combustion sparks, no smoking, no with combustible and

m.p. –218.4 °Crespiratory tract. of other substances. contact with flammable reducing materials,

b.p. –183 °C. Heating will cause rise substances. causing fire and explosion

in pressure of container hazard. Reacts with oils,

with risk of bursting. greases, hydrogen, and

flammable liquids, solids

and gases.

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 160 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Perchloric acid Colourless liquid; Corrosive; causes severe Powerful oxidizing Avoid breathing vapour Combustible materials Powerful oxidizing

HClO

miscible with water.burns to eyes and skin agent. Not combustible and other exposure; wear and reducing agents: agent; may form

and if ingested. Vapour but enhances protective clothing acetic anhydride, explosive products

is corrosive to eyes, skin, combustion of other including nitrile gloves, bismuth and its alloys, if in contact with

and respiratory tract. substances. eye and face protection. alcohol, metal, paper,many inorganic and

Inhalation of vapours With hot solutions work wood and other organic organic materials;

may cause lung in fume cupboard or hood. materials. contaminated wood-

oedema. en floors, benches,

etc. May explode on

percussion.

Phenol Colourless or pale Substance and vapours Flashpoint 80 °CDo not breathe vapour; Reacts with oxidants

OH pink crystals with are corrosive to eyes, flammable range use respiratory protection. causing fire and

characteristic odour; skin and respiratory 1.7–6%. Avoid eye and skin contact. explosion hazard.

m.p. 41 °Ctract causing severe Work in fume cupboard.

b.p. 182 °C; burns; absorbed through Wear nitrile gloves and

soluble in water. skin. Central nervous eye protection. In case of

system disturbance, contact with eyes, rinse

coma. Kidney and liver immediately with water

damage. Symptoms and seek medical advice;

include abdominal pain, in case of contact with

vomiting, diarrhoea, skin skin, remove any

irritation, eye pain. contaminated clothing

Prolonged contact with and swab the

dilute solutions may contaminated area with

cause dermatitis. glycerol, polyethylene

glycol 300 or a mixture

of liquid polyethylene

glycol (70%) and methyl-

ated spirit (30%) and then

flush with water.

• 161 •

Phosphoric Colourless viscous Corrosive; causes Attacks many metals In case of contact with

acid liquid or hygroscopic burns to the skin and producing hydrogen. eyes, rinse with water and

white crystals; eyes. Gives off toxic fumes obtain medical advice.

m.p. 42 °Cin a fire. Wear nitrile gloves and

decomposes below eye protection.

boiling point at 213 °C;

soluble in water.

Phosphorus Hygroscopic white Corrosive to the eyes, Not combustible but Work with local exhaust Solution in water is a

pentoxide crystals or powder; skin, respiratory tract, enhances combustion protection. Wear strong acid; reacts

m.p. 340 °C, sublimation leading to sore throat, of other substances. protective gloves and violently with bases and

point 360 °C. cough, burning Many reactions may clothing, face shield, or is corrosive. Reacts

sensation, shortness of cause fire or explosion. eye protection in violently with perchloric

breath; skin burns, pain, Gives off irritating or combination with acid causing fire and

blisters, eye burns. toxic fumes (or gases) respiratory protection. explosion hazard. Reacts

Inhalation may cause in a fire. violently with water

lung oedema. Ingestion forming phosphoric acid.

may cause abdominal Attacks many metals in

cramps, burning presence of water.

sensation, diarrhoea,

sore throat, vomiting.

Picric acid Yellow crystals Toxic by ingestion, Explosive when dry. Keep moistened with Forms salts with many Yellow skin stains.

(NO

)

OH moistened with water inhalation or skin water at all times or use metals which are more

2,4,6- or dissolved in alcohol; contact. Ingestion may only in alcoholic solution. explosive than the acid

Trinitrophenol m.p. 122 °C; result in headache, itself. In contact with

slightly soluble in water.nausea. Irritation of concrete may form

eyes. calcium picrate, which is

a friction-sensitive

explosive. May react

vigorously with reducing

agents.

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 162 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Potassium White flakes, powder,Corrosive to In case of contact with Reacts violently with Attacks some

hydroxide pellets or sticks; respiratory system, eyes, rinse immediately acids and with nitro- metals (aluminium,

KOH m.p. 360 °Ceyes and skin; with water and seek benzene and many zinc, tin) in the

b.p. 1320 °C; inhalation of dust medical advice; in case other detergents. presence of

very soluble in water.causes lung oedema. of contact with skin, wash Evolves large quantity moisture.

immediately; remove of heat when mixed

contaminated clothing. with water; store in a

Wear rubber or plastic well-sealed container.

gloves and eye protection

even for dilute solutions.

Potassium Purple crystals; Corrosive if swallowed Powerful oxidizing Wear protective clothing, Reacts violently or

permanganate m.p. 240 °Cor if dust is inhaled. agent; may ignite eye protection and explosively if mixed with

KMnO

(decomposes); Extreme irritation of combustible materials. particulate respirator if a wide variety of

readily soluble in water.eyes and respiratory dust is produced. inorganic and organic

tract, inhalation of dust compounds or powdered

may cause lung oedema. metals.

Potassium White deliquescent Toxic by ingestion and Wear protective

tellurite crystals; very soluble inhalation of dust. clothing.

TeO

in water. Irritation of skin and eyes.

Propan-2-ol Colourless liquid with Irritation of eyes and Highly flammable; Keep container tightly Can react vigorously 70–85% propan-2-

(CH

)

CHOH alcoholic odour; respiratory tract. May flashpoint 112 °Cclosed; keep away from with oxidizers to form ol in water used as

Isopropanol m.p. –89 °Caffect central nervous flammable range ignition sources. Work in unstable peroxides on a disinfectant spray

b.p 82 °C; system causing 2.3–12.7%. fume cupboard. Wear prolonged exposure to remains a

miscible with water.headache, dizziness, nitrile gloves and eye air and light. flammable hazard

nausea, vomiting and protection. and should not be

coma. used near ignition

sources.

• 163 •

Pyridine Colourless liquid with Affects central nervous Highly flammable; Work with ventilation, Reacts violently with

Ncharacteristic odour; system causing dizzi- flashpoint 20 °Clocal exhaust or strong oxidants and

m.p. 42 °Cness, headache, nausea, explosive limits, respiratory protection; strong acids.

b.p. 115 °C. shortness of breath, 1.8–12.4%. wear gloves and

unconsciousness. May Gives off irritating or protective clothing.

be absorbed through skin toxic fumes (or gases)

causing redness and in a fire. Vapour/

burning sensation. In- mixtures are explosive.

gestion causes abdominal

pain, diarrhoea, vomiting,

weakness. Repeated

exposure causes liver

and kidney effects.

Selenium Odourless solid in Irritation of skin and Combustible. Gives Prevent dispersion of Reacts violently with oxi-

Se various forms, dark eye. Inhalation of dust off irritating or toxic dust. Observe strict dants and strong acids.

red-brown to bluish- may cause lung oedema. fumes (or gases) in hygiene. Work with local Reacts with water at 50 °C

black amorphous solid Repeated exposure may a fire. exhaust. Wear protective forming flammable

or red transparent cause loss of nails, gloves, clothing, and hydrogen and selenious

crystals or metallic grey gastrointestinal effects. safety spectacles. acids. Reacts with incan-

to black crystals; descence on gentle

m.p. 170–217 °Cheating with phosphorus

b.p. 685 °C. and metals such as nickel,

potassium, platinum,

sodium and zinc.

Silver White metal, turns dark Inhalation of high Not combustible Work with local exhaust. Incompatible with

Ag on exposure to ozone, amounts of metallic except as powder. Wear protective gloves acetylene, ammonium

hydrogen sulfide or silver vapours may cause and safety spectacles or compounds, oxalic acid

sulfur; lung damage with eye protection in and tartaric acid.

m.p. 962 °Cpulmonary oedema. May combination with

b.p. 2212 °C. cause a grey-blue discol- respiratory protection for

oration of the eyes, nose, powder or fume.

throat and skin on long-

term or repeated

exposure (argyria).

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

• 164 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Silver nitrate White crystals; May cause severe Not combustible but Prevent dispersion of Ammoniacal solutions

AgNO

m.p. 212 °Cirritation and burns to enhances combustion dust. Observe strict can precipitate explosive

b.p. 444 °C; eyes and skin. Corrosive of other substances. hygiene. Wear protective silver nitrite in the

soluble in water. by ingestion. May cause rubber or plastic gloves, presence of base or

a red-blue discoloration and face shield or eye glucose. Can form

of the skin on long-term protection in combination explosive products with

or repeated exposure with respiratory protection. ethanol and may cause

(argyria). In case of contact with explosive polymerization

eyes, rinse with water with acrylonitrile. May

and seek medical advice. cause ignition of explosion

if mixed with charcoal,

magnesium, phosphorus

or sulfur.

Sodium azide Colourless crystalline Very toxic by ingestion, Decomposes In case of contact with Explosive reactions with

Na solid; inhalation and skin explosively when skin, wash immediately.bromine, carbon disulfide

m.p. 300 °C; contact; may cause heated above its melt- Do not inhale dust. or chromyl chloride. Solid

soluble in water. burns. Dust and ing point. Gives off Wear rubber or plastic reacts with heavy metals

solution irritate eyes and toxic fumes when gloves and eye including copper, lead and

skin; may be absorbed heated; do not use protection. mercury to form explosive

through skin. water to extinguish metal azide salts. On

fires. contact with acid, develops

highly toxic and explosive

gas.

Sodium Colourless, white Toxic by ingestion and Wear protective clothing. Oxidizing agents.

biselenite crystalline powder; inhalation of dust;

NaHSeO

soluble in water. possible danger of

cumulative effects.

Experimental teratogen.

Prolonged skin contact

may cause dermatitis.

• 165 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Sodium cyanide White crystalline Extremely toxic by May give off toxic Do not inhale dust; use Liberates extremely Treat spillage of

NaCN powder with almond ingestion, inhalation fumes in a fire. respiratory protection. toxic hydrogen cyanide solutions with

odour; and skin contact; Avoid eye and skin contact; (HCN) gas on contact bleaching powder

m.p. 563 °Cseverely irritating to in case of contact with with acids or with water (sodium hypo-

b.p. 1496 °C; eyes. May be absorbed skin, wash immediately containing dissolved chlorite) and leave

very soluble in water. through skin. Repeated with water and remove carbon dioxide. Can for 24 h. Sweep up

exposure may affect contaminated clothing. form explosive mixtures solid spills carefully

thyroid. Wear chemical-grade with nitrites. and add to water

goggles and rubber or containing

plastic gloves. Keep in a bleaching powder;

securely locked, ventilated leave for 24 h

store. before discarding.

Keep cyanide anti-

dote kit available in

the laboratory.

Sodium Colourless flakes, Solid and concentrated Not combustible. In case of contact with Evolves large quantity Store in well-sealed

hydroxide powder, pellets or solute. Inhalation of Contact with moisture eyes rinse immediately of heat when mixed with container in dry

NaOH sticks; dust causes damage to or water may generate and seek medical advice; water. Reacts vigorously place.

m.p. 318 °Crespiratory tract, lung sufficient heat to in case of contact with with chloroform-

b.p. 1390 °C; oedema. Corrosive by ignite combustible skin wash immediately methanol mixtures and

soluble in water. ingestion. Dilute substances. with water, remove with strong acids.

solutions irritating to contaminated clothing.

eyes or may cause severe Wear rubber or plastic

damage if eye contact is gloves and eye protection

prolonged. even with dilute solutions.

• 166 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Sodium Colourless or pale Corrosive to eyes and Strong oxidant. May In case of contact with Liberates highly toxic Gradually loses

hypochlorite yellow solution with skin; corrosive by give off toxic fumes eyes, rinse immediately gas in contact with acids. chlorine during

solution chlorine odour; ingestion and to in a fire. with water and seek Can react vigorously storage; dilute

(10–14% miscible with water.respiratory tract; medical advice; in case of with combustible and solutions used as

available inhalation may cause contact with skin, wash reducing compounds. disinfectant rapidly

chlorine) lung oedema. Repeated immediately. Do not inhale May react with nitrogen deteriorate. Store

NaOCl exposure may cause vapour; use respiratory compounds to form away from acids in

skin sensitization. protection. Work in well- explosive N-chloro- a dark, cool, well-

ventilated area. Wear compounds; may react ventilated area.

rubber or plastic gloves violently with methanol.

and chemical-grade eye

protection.

Sulfuric acid Colourless, odourless Concentrated solution May give off toxic In case of contact with Is a powerful oxidizing Localized boiling

viscous liquid; (15%) corrosive, causes fumes in a fire. Not eyes rinse immediately desiccant and reacts may occur if

m.p. 10°Csevere burns; mist and combustible. Many and seek medical advice; violently with many concentrated acid

b.p. (decomposes) vapour highly corrosive reactions may cause in case of contact with reagents including is added to water.

340 °C. by inhalation; dilute fire or explosion. skin wash immediately organic nitro compounds,

solutions irritating to Dilution with water remove contaminated potassium permanganate,

eyes and skin; cause generates heat and clothing. Wear nitrile alkali metals and per-

burns and dermatitis. spattering or boiling gloves, eye and face chlorates, combustible

may occur. Always protection. No contact materials, oxidizers,

add acid to water, with flammable amines, bases, water,

never add water to substances.excess heat and most

acid.metals.

• 167 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Tetrahydrofuran Colourless liquid, with Central nervous system Highly flammable; Work with ventilation, Reacts violently with

Ocharacteristic odour; depressant causing may form explosive local exhaust or strong oxidants, strong

Diethylene oxide m.p. –108.5 °Cnarcosis. Eye, skin and peroxides; respiratory protection, bases and some metal

Tetramethylene b.p. 66 °C. respiratory irritation. flashpoint –14 °C. protective gloves, safety halide, causing fire and

oxide Water may be ineffect- spectacles. explosion hazard. Attacks

ive to fight fires some forms of plastics,

involving tetrahydro- rubber and coatings.

furan, but it can be Tetrahydrofuran may

used to cool fire- polymerize in the presence

exposed containers. of cationic initiators.

Refluxing with calcium

hydroxide can cause

explosions.

Thallium acetate White deliquescent Extremely toxic by Keep container tightly

TlC

crystals; ingestion with possible closed. Work in fume

m.p. 110 °C; cumulative effects. cupboard, hood or

very soluble in water.Affects nervous and with exhaust ventilation.

cardiovascular systems. Wear protective clothing

Harmful through eye and including dust respirator,

skin contact. chemical-grade goggles,

rubber or plastic gloves,

eye protection.

-Tolidine Colourless crystals; Harmful by contact with Combustible. Gives Avoid contact; wear eye Oxidizing agents.

-(3CH

)- m.p. 131 °Cskin or ingestion. Dust off irritating or toxic protection and gloves.

(4NH

))

b.p. 200 °C; irritates respiratory tract fumes (or gases) in

3,3'-Dimethyl- poorly soluble in water.and eyes. Probably a a fire.

(1,1'-biphenyl)- human carcinogen.

4,4'-diamine

• 168 •

LABORATORY BIOSAFETY MANUAL

CHEMICAL PHYSICAL PROPERTIES HEALTH HAZARDS FIRE HAZARDS SAFETY PRECAUTIONS INCOMPATIBLE CHEMICALS OTHER HAZARDS

Toluene Colourless liquid with Central nervous Highly flammable; Keep container tightly Can react with strong

characteristic odour; system depressant. vapour may cause closed; keep away from acids, alkalis and

Methylbenzene m.p. –95 °CIrritation of eyes, flash fire; ignition sources; earth/ oxidizers.

b.p. 111 °C; mucous membranes, flashpoint 4 °C(ground) containers to

not miscible with water.skin. Repeated exposure flammable range prevent static electrical

may cause toxicity in 1.4–7%. discharge. Do not inhale

human reproduction or Extinguishing media vapour; use respiratory

development. for a small fire: dry protection. Work in fume

chemicals, carbon cupboard or well-ventilated

dioxide, foam, water area. Wear nitrile gloves.

fog or inert gas

(nitrogen).

Trichloroacetic White hygroscopic Corrosive; causes Not combustible. May Avoid contact with eyes Violent reaction with Store in a dry place.

acid crystals with pungent severe burns to eyes, give off toxic fumes and skin; wear rubber or copper/dimethyl Concentrated

CCl

COOH odour; skin, respiratory tract. in fire. plastic gloves and sulfoxide mixtures and aqueous solutions

m.p. 58 °Cchemical-grade goggles on contact with bases, may decompose

b.p. 197.5 °C; or face shield in strong oxidizing agents violently.

soluble in water, ethanol, combination with and metals such as iron,

diethylether. respiratory protection. In zinc, aluminium.

case of contact with eyes,

rinse immediately and

seek medical advice.

• 169 •

ANNEX 5. CHEMICALS: HAZARDS AND PRECAUTIONS

Trichloro- Colourless liquid, Irritation of eyes, skin; Combustible under Work with ventilation, On contact with hot

ethylene characteristic odour; prolonged exposure specific conditions. local exhaust. Wear surfaces or flames,

CHClCCl

m.p. –73 °Cmay cause dermatitis gloves, safety spectacles decomposes forming

b.p. 87 °C. and affect the central or other eye protection toxic and corrosive gases

nervous system resulting in combination with (phosgene, hydrogen

in loss of memory. May respiratory protection. chloride). Decomposes

affect liver and kidneys. on contact with strong

Probably a human alkali producing dichloro-

carcinogen. acetylene; reacts violently

with metal powders such

as aluminium, barium,

magnesium and titanium;

slowly decomposed by

light in the presence of

moisture, with formation

of hydrochloric acid.

Xylene (mixed Colourless liquid with May affect central Flammable liquid; Avoid contact with eyes. May contain

isomers) aromatic odour; nervous system flashpoint 27–32 °C. Wear nitrile gloves and ethylbenzene as an

(CH

)

m.p. –95 to –13 °Cresulting in headache, eye protection. Keep impurity.

Dimethyl- b.p. 136–145 °C; dizziness, fatigue and container tightly closed; Ethylbenzene is a

benzene insoluble in water. nausea. Liquid and keep away from ignition possible human

vapour irritate eyes, skin, sources. carcinogen.

mucous membranes,

respiratory tract. Harmful

if ingested. Prolonged

skin contact may defat

the skin. Non-specific

neurological impairment.

Exposure may enhance

hearing damage caused

by exposure to noise.

Animal tests suggest

toxicity to human repro-

duction or development.

Index

• 170 •

abrasions 79

access

animal facilities 29, 30, 31

laboratory 9–10, 21, 26

accidents 11

equipment-related 144

Biosafety Laboratory Manual Biosafety7

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