esinelidguimterin World Health Organization 2003This publication is not a formal publication of the World Health Organization (WHO), and all rights arereserved by the Organization. The publication may, however, be freely reviewed, abstracted, reproducedand translated, in part or in whole, but not for sale or for use in conjunction with commercial purposes.The views expressed in publications by named authors are solely the responsibility of those authors.The designations employed and the presentation of the material in this publication, including tables andmaps, do not imply the expression of any opinion whatsoever on the part of the secretariat of the WorldHealth 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 approximateborder 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 areendorsed or recommended by WHO in preference to others of a similar nature that are not mentioned.Errors and omissions excepted, the names of proprietary products are distinguished by initial capitalletters.

WHO/CDS/CSR/LYO/2003.4ContentsForeword . vAcknowledgements . vii1.General principles. 1PART I GuidelinesRisk assessment. 7Specimens for which there is limited information . 7Risk assessment and genetically modified microorganisms . 83.Basic laboratories – Biosafety Levels 1 and 2. 10Code of practice. 10Laboratory design and facilities . 11Laboratory equipment . 13Health and medical surveillance . 13Training . 14Waste handling . 14Chemical, fire, electrical and radiation safety . 164.The containment laboratory – Biosafety Level 3 . 17Code of practice. 17Laboratory design and facilities . 17Laboratory equipment . 18Health and medical surveillance . 185.The maximum containment laboratory – Biosafety Level 4 . 20Laboratory design and facilities . 20Laboratory biosafety manual . 216.Laboratory animal facilities . 22Animal facility – Biosafety Level 1 . 22Animal facility – Biosafety Level 2 . 23Animal facility – Biosafety Level 3 . 23Animal facility – Biosafety Level 4 . 24Invertebrates . 24interimguidelines2.PART II Laboratory equipment7.Biological safety cabinets. 29Class I biological safety cabinet. 30Class II biological safety cabinets. 30Class III biological safety cabinet. 32Biological safety cabinet air connections . 33Selection of a biological safety cabinet. 33Using biological safety cabinets in the laboratory. 348.Equipment-related hazards. 37Equipment that may create a hazard. 37i

Laboratory Biosafety Manual, 2nd revised edition9.WHO/CDS/CSR/LYO/2003.4Equipment designed to reduce biological hazards. 39Negative-pressure flexible-film isolators . 40Pipetting aids . 40Homogenizers, shakers, blenders and sonicators. 41Disposable transfer loops . 41Microincinerators. 41Personal protective clothing and equipment. 41PART III Good microbiological techniqueidelines10. Safe laboratory techniques. 45Safe handling of specimens in the laboratory. 45Use of pipettes and pipetting aids. 45Avoiding the dispersal of infectious materials. 46Use of biological safety cabinets . 46Avoiding ingestion of infectious materials and contact with skin and eyes . 46Avoiding injection of infectious materials . 47Separation of serum . 47Use of centrifuges . 47Use of homogenizers, shakers, blenders and sonicators. 48Use of tissue grinders . 48Care and use of refrigerators and freezers. 48Opening of ampoules containing lyophilized infectious materials . 48Storage of ampoules containing infectious materials . 49Special precautions with blood and other body fluids, tissues and excreta . 49Precautions with materials that may contain prions . 50imgu11. Biosafety and recombinant DNA technology. 52Biological expression systems. 52Properties of the donor organism and cloned DNA . 52Viral vectors for gene transfer. 53Transgenic and “knock-out” animals. 53Transgenic plants. 53Conclusions . 53ter12. Transport of infectious substances. 55in13. Contingency plans and emergency procedures. 56Contingency plan . 56Emergency procedures for microbiological laboratories. 5614. Disinfection and sterilization . 59Definitions . 59Precleaning and cleaning laboratory materials. 59Chemical germicides. 60Local environmental decontamination . 63Decontamination of biological safety cabinets. 63Hand-washing/hand decontamination . 64Heat disinfection and sterilization . 64Incineration . 66Disposal . 66Decontamination of prion-containing materials . 66Summary. 66PART IV Chemical, fire and electrical safety15. Hazardous chemicals . 69Definitions and classifications. 69Routes of exposure. 69Storage of chemicals . 69ii

WHO/CDS/CSR/LYO/2003.4ContentsGeneral rules . 70Toxic effects of chemicals. 70Explosive chemicals. 72Chemical spillage. 72Compressed and liquefied gases . 7316. Fire in the laboratory . 7417. Electrical hazards . 75PART V Safety organization and training18. The biosafety officer and safety committee. 77Biosafety officer . 77Safety committee . 78General organization. 78ines19. Safety rules for support staff. 79Engineering and building maintenance services . 79Cleaning (domestic) services. 79Safety rules for domestic and cleaning staff. 79guidel20. Training programmes . 81Basic course: Good laboratory practice (GLP) . 82Module 1 (the core module): Good microbiological technique (GMT). 83Module 2: The safe laboratory environment . 83Module 3: GLP for support staff. 84Module 4: GLP for safety staff . 84Module 5: GLP for specialist staff who handle microorganisms in Risk Groups 3 and 4 . 85interim21. Safety checklist . 87Laboratory premises . 89Storage facilities. 89Sanitation and staff facilities . 89Heating and ventilation . 89Lighting . 90Services . 90Security . 90Fire prevention . 90Flammable liquid storage. 90Electrical hazards. 91Compressed and liquefied gases . 91Personal protection . 91Health and safety of staff . 91Laboratory equipment . 92Infectious materials . 92Chemicals and radioactive substances . 92References . 94ANNEX 1 Immunization of staff . 98ANNEX 2 WHO Biosafety Collaborating Centres. 99iii


WHO/CDS/CSR/LYO/2003.4ForewordimguidelinesThe World Health Organization (WHO) has long recognized that safety and, in particular, biological safetyare important international issues. This specialized agency of the United Nations published the firstedition of its Laboratory biosafety manual in 1983. The manual encourages countries to prepare specificcodes of practice for the safe handling of pathogenic microorganisms in laboratories within theirgeographical borders, and provides expert guidance for developing such codes of practice.This web-based revision of the second edition is the first step in the preparation of a third edition,planned for publication in 2003. The new publication will combine the Laboratory biosafety manual, 2nd1ed. (revised) and the contents of the document entitled Safety in health-care laboratories .The authors and editor of this revised second edition continue the now-established WHO tradition ofpromoting health and safety in microbiological laboratories in the international community. While thetechnical content affirms the guidance of previous editions, there are helpful changes in the organizationof the text and valuable new material is included. The importance of personal responsibility for safelaboratory activities is stressed throughout the manual. A safe and healthful laboratory environment is theproduct of individuals who are well trained and technically proficient in safe practices, and shareresponsibility for their own safety and for the safety of their colleagues, their communities and theenvironment. Personal responsibility also involves the practice of assessing risks prior to the conduct ofactivities that involve new protocols or new pathogens. Two new chapters address risk assessment andrecombinant DNA technology. These timely additions provide thoughtful and concise guidance forassessing risks in the contemporary microbiology laboratory.A safe and healthful laboratory environment is also the product of responsible institutional leadership.National codes of practice foster and promote good institutional leadership in biosafety. The revisedsecond edition of the WHO Laboratory biosafety manual is, like the previous editions, a helpful referenceand guide to nations that accept the challenge to develop national codes of practice.interW. Emmett Barkley, PhDDirector, Office of Laboratory SafetyHoward Hughes Medical InstituteChevy Chase, MD, USA1Safety in health-care laboratories. Geneva, World Health Organization, 1999 (unpublished document WHO/LAB/97.1). Obtainableon request from Department of Vaccines and Other Biologicals, World Health Organization, 1211 Geneva 27, Switzerland;


WHO/CDS/CSR/LYO/2003.4AcknowledgementsThe development of this revised second edition of the Laboratory biosafety manual has been madepossible through the contributions of the following, whose expertise is gratefully acknowledged:interimguidelinesDr Ingegerd Kallings, Swedish Institute of Infectious Disease Control, Stockholm, SwedenMs Mary Ellen Kennedy, Consultant in Biosafety, Ashton, Ontario, Canada (Technical editor)Ms Margery Kennett, Victorian Infectious Diseases Reference Laboratory, North Melbourne, AustraliaThe late Dr Richard Knudsen, Office of Health and Safety, Centers for Disease Control andPrevention, Atlanta, GA, USADr Nicoletta Previsani, Biosafety Programme, World Health Organization, Geneva, SwitzerlandDr Jonathan Richmond, Office of Health and Safety, Centers for Disease Control and Prevention,Atlanta, GA, USADr Syed A. Sattar, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, CanadaDr Deborah E. Wilson, Division of Safety, National Institutes of Health, Washington, DC, USADr Riccardo Wittek, Institute of Animal Biology, University of Lausanne, Lausanne, Switzerlandvii


WHO/CDS/CSR/LYO/2003.41. General principlesThroughout this manual, references are made to the relative hazards of infective microorganisms by riskgroup (WHO Risk Groups 1, 2, 3 and 4). This risk group classification is to be used for laboratory workonly. Laboratories are designated according to their design features, construction and containmentfacilities as basic – Biosafety Level 1, basic – Biosafety Level 2, containment – Biosafety Level 3 andmaximum containment – Biosafety Level 4. Table 1 describes the risk groups, Table 2 relates them to thelaboratory designations, and Table 3 summarizes the requirements at the four biosafety levels.idRisk Group 2 (moderate individual risk, low community risk)elA microorganism that is unlikely to cause human or animal disease.esRisk Group 1 (no or very low individual and community risk)ainTable 1. Classification of infective microorganisms by risk groupimguA pathogen that can cause human or animal disease but is unlikely to be a serious hazard tolaboratory workers, the community, livestock or the environment. Laboratory exposures may causeserious infection, but effective treatment and preventive measures are available and the risk of spreadof infection is limited.Risk Group 3 (high individual risk, low community risk)interA pathogen that usually causes serious human or animal disease but does not ordinarily spread fromone 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 transmittedfrom one individual to another, directly or indirectly. Effective treatment and preventive measures arenot usually available.aThe concept and classification of risk groups are being reevaluated and will be addressed in the third edition of the Laboratorybiosafety manual.Table 2. Relation of risk groups to biosafety levels, practices and equipmentRiskGroup12Biosafety LevelLaboratory typeLaboratory practicesSafety equipmentBasic – BiosafetyLevel 1Basic teaching,researchGMTNone; open benchworkBasic – BiosafetyLevel 2Primary healthservices;diagnostic,researchGMT plus protectiveclothing, biohazardsignOpen bench plusBSC for potentialaerosols1

Laboratory Biosafety Manual, 2nd revised editionRiskGroup34WHO/CDS/CSR/LYO/2003.4Biosafety LevelLaboratory typeLaboratory practicesSafety equipmentContainment – BiosafetyLevel 3Specialdiagnostic,researchAs Level 2 plus specialclothing, controlledaccess, directional airflowBSC and/or otherprimary devices forall activitiesMaximum containment –Biosafety Level 4Dangerouspathogen unitsAs Level 3 plus airlockentry, shower exit,special waste disposalClass III BSC, orpositive pressuresuits in conjunctionwith Class II BSCs,double-endedautoclave (throughthe wall), filtered airesBSC, biological safety cabinet; GMT, good microbiological techniqueelim–id–Pathogenicity of the organism.Mode of transmission and host range of the organism. These may be influenced by existing levels ofimmunity in the local population, density and movement of the host population, presence ofappropriate vectors, and standards of environmental hygiene.Local availability of effective preventive measures. These may include: prophylaxis by immunizationor administration of antisera (passive immunization); sanitary measures, e.g. food and water hygiene;control of animal reservoirs or arthropod vectors.Local availability of effective treatment. This includes passive immunization, postexposurevaccination, and use of antimicrobials, antivirals and chemotherapeutic agents, and should take intoconsideration the possibility of the emergence of drug resistant––inCountries (regions) should draw up a national (regional) classification of microorganisms, by riskgroup, based on the following factors.terTable 3. Summary of biosafety level requirementsIsolation of laboratoryinBiosafety levelRoom sealable for lation:–inward air flowNoDesirableYesYes–mechanical via building systemNoDesirableYesNo–mechanical, independentNoDesirableYesYes–HEPA filtered air exhaustNoNoDesirableYesDouble-door entryNoNoYesYesAirlockNoNoNoYesAirlock with showerNoNoNoYesAnteroomNoNoYesNoAnteroom with showerNoNoDesirableNoEffluent treatmentNoNoDesirableYes2

WHO/CDS/CSR/LYO/2003.4General principlesBiosafety level1234Autoclave:–on siteYesYesYesYes–in laboratory sBiological safety cabinets:Class INoOptionalYesNo–Class IINoDesirableYesYes, inconjunctionwith suitlaboratories–Class IllNoNoDesirableYes, inconjunctionwith cabinetlaboratorieselines–interimguidIn assessing the various criteria for classification, it is important to take into account conditionsprevailing in the geographical area in which the microorganisms are handled.In the preparation of classification lists it is recommended that, where appropriate, some additionalinformation is given about the advisability of the use of personal protective equipment and primarycontainment devices (e.g. biological safety cabinets). Consideration must also be given to enhancingbiosafety practices and procedures and general containment levels for organisms known to be multidrugresistant and in cases where high volumes or concentrations of an agent may be used. Examples ofclassification lists are available from the Centers for Disease Control and Prevention (CDC) and theNational Institute of Health (NIH), USA (1), and the European Union (EU) (2).3


PART IinterimguidelinesGuidelines5


WHO/CDS/CSR/LYO/2003.42. Risk assessment–idguimter––pathogenicity of the agent and infectious doseconsideration of the outcome of exposurenatural route of infectionother routes of infection, resulting from laboratory manipulations (parenteral, airborne, ingestion)stability of the agent in the environmentconcentration of the agent and volume of concentrated material to be manipulatedpresence of a suitable host (human or animal)information available from animal studies and reports of laboratory-acquired infections or clinicalreportslaboratory activity planned (concentration, sonication, aerosolization, centrifugation, etc.)any genetic manipulation of the organism that may extend the host range of the agent or alter theagent’s sensitivity to known, effective treatment regimens (see Risk assessment and geneticallymodified microorganisms, below)local availability of effective prophylaxis or ther