Understanding BiosafetyHazard control, hygiene and infection protection in the lab

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Ensure your personal safetyUnderstand how to stay safe while handling pathogens.

Know the risks, ensure the correct controls are in place and select the appropriate Personal Protective Equipment (PPE) to keep you safe, healthy and protected in your laboratory.

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What are biohazards?Biosafety terminology varies throughout the globe.

A pathogen is a bacterium, virus, or other micro-organism that can cause disease.

A biological material is any item or substance which has been sampled or derived from a biological source, including whole animals or other organisms and parts or tissues derived from these or samples taken from a habitat or ecosystem.

A biological agent is a micro-organism, cell culture, or human endoparasite, whether or not genetically modified, which may cause infection, allergy, toxicity or otherwise create a hazard to human health.

Genetic Modification, also referred to as GM, genetic engineering or recombinant DNA technology, is the creation of new combinations of genetic materials within a viable micro-organism (GMM), animal or plant (GMO, transgenic organism).

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Micro-organisms include:

• Bacteria• Viruses• Fungi• Microscopicendoparasites

Other biohazards include:

• Workwithhumanorprimatetissues• Biologictoxins• Cellsorcellculture• RecombinantDNA• Transgenicplantsoranimals• Humangenetherapytransfer• Zoonoticdiseases

Bacteria Endoparasite

Virus

Fungus

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Biological risk classificationPathogens are classified into one of four Biosafety Levels (orHazardGroups)basedontheirabilitytoinfecthealthyindividuals and spread to the community.1

1.WorldHealthOrganization.Laboratorybiosafetymanual.3rded.Geneva;2004.

BSL-4 High Risk Pathogen

Low Risk Pathogen

BSL-3

BSL-2

BSL-1

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Pathogen classificationClassificationsofbiologicalagentsareusedinriskassessmentsfor work with biological agents and the application of appropriate control measures. Each country classifies the agents in that country by risk group based on pathogenicity of the organism, modesoftransmissionandhostrangeoftheorganism.Thesemay be influenced by existing levels of immunity, density and movement of host population presence of appropriate vectors and standards of environmental hygiene.

European Union (EU)• EuropeanDirective2000/54/EC,AnnexIIICommunity Classificationsofbiologicalagents: www.biosafety.be/PDF/2000_54.pdf

UK• ApprovedListofbiologicalagentsHSE/HSC2004 (basedonAnnexIII):www.hse.gov.uk/pubns/misc208.pdf

USA • BiosafetyinMicrobiologicalandBiomedicalLaboratories, 4thEdition,1999,CDC/NIHguidelines www.cmrr.umn.edu/safety/Biosafety.pdf

• NIHRecombinantDNAGuidelines(USA,2002)April2002. AppendixB: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html

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Exposure potentialTherearethreewaysinwhichyoumightbeexposed topathogensatwork:

1. Biological laboratory work

2.Incidentalexposureto contaminants e.g. farming, refuse, sewage

3.Incidentalinfection from other colleagues

Only the first two are covered by health and safety legislation.

ChainofinfectionThechainofinfectionrepresentstheprocessbywhichinfectiontakesoverthebodyandcausesharm.So,bybreakingthechain,we regain control.

SourceThefourmainsourcesofworkplaceinfectionare:

1. Blood and other body fluids (eg saliva) - human bodies, animal carcases and raw meat

2.Humanoranimalwasteproducts-faeces,urineandvomit3.Respiratorydischarges-coughsandsneezes4.Skin–directcontact

Susceptible

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TransmissionTobecomeinfected,themicro-organismmust travel from the sourceintothehostbysomemeans.Routesofentryinclude:

1. Putting contaminated objects or fingers in the mouth

2. Breathing in aerosols or droplets

3.Splashesofbloodorfluidsinto eyes, nose or mouth

4. Direct contact with broken skin

5.Skin-penetratinginjuries

Host susceptibilityOur final line of defence against infection is our immune system.

Individualimmunitylevelsareinfluencedby:

• pre-existingillnesses• previousinfection• naturalimmunity• immunisation

Ifourimmunesystemiscompromisedinanyway,wearemorelikely to succumb to infection and carrying out less hazardous tasks may be advisable. Boosting immunity can also provide an effective control measure.

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4.

5.

3.2.1.

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Biohazard control measuresForbasiccontrolspleaserefertothe‘Understandinglaboratory risk management’ miniguide. Engineering controls

• Designeasy-to-cleanfacilities• Selecteasy-to-cleanequipment• Airflowcontrol-biosafetycabinets•Manageandtreatwatersystems• Contaminationcontrol: o appropriate PPE o surface disinfection o autoclave

• Safetyequipment: o eyewash o safety shower o fireblankets/extinguishers

Equipment to reduce biological hazards• Negative-pressureflexible-filmisolators• Pipettingaids• Homogenizers,shakers,blendersandsonicators• Disposabletransferloops•Microincinerators

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Reportable infection incidents are those that: • resultinsignificantmorbidityormortality• involvehighlyvirulentorganisms • arereadilytransmissible • requirecontrolmeasuresthat have an impact on other employees

Footnote2.WorldHealthOrganization.Laboratory biosafety manual. 3rded.Geneva;2004

Special biosafety considerations• Biosafetyprogram

• Goodmicrobiologicaltechnique2

• Exposurecontrolplanningfor:

o Bloodborne pathogens

o AerosolTransmissibleDiseases(ATD)

• Respiratoryprotection

• Immunisation

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Working with biohazards

Footnote3.http://www.who.int/csr/resources/publications/biosafety/Labbiosafety.pdfWorldHealthOrganizationLABORATORYBIOSAFETYMANUAL.2003

FacilitiesLaboratories are designated according to their design features, construction and containment facilities as Biosafety levels 1-4. Thetablebelowindicatesthedifferencesbetweeneach.3

Risk BiosafetyLevel Definitions Laboratory Type Laboratory Practices

1Basic BiosafetyLevel 1

No or very low individual and community risk A micro-organism that is unlikely to cause human or animal disease

Basic teaching research GMT/GLP

2Basic BiosafetyLevel 2

Moderate individual risk, low community risk A pathogen that can cause human or animal disease but unlikely to be a serious hazard to laboratory workers. Laboratory exposures may cause serious infection, but effective treatment and preventative measures are available and the risk of spread of infection

Primary health services diagnostic research GMT plus protective clothing as part of GLP

3Containment BiosafetyLevel 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 preventative measures are available

Special diagnosticresearch

As Level 2 plus special clothing controlled access, directional air flow, biological safety cabinets

4MaximumContainment BiosafetyLevel 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 preventative measures are not usually available

Dangerous pathogen units Basic teaching research

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Handling•WearappropriatePersonalProtectiveEquipment(PPE)

• SelectaglovewiththeappropriatelevelofCEcertificationto address your application risk and performance requirements –See Understanding glove certification guide

• SterilisetoolsintheblueflameofaBunsenburner

•Wipedownyourworkingsurfacebeforeandafterwith70% ethanol

• Ensureappropriatetransportationorwastemanagement equipment is to hand

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1. Risk assessment

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10. Regular evaluation and review

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A biological safety cabinet creates a force field of air that washes any airborne contaminants towards a filter. A glass window shields against splashes.

When using a biosafety cabinet:

• TurnontheUVlightpriortoworktosterilisetheinteriorofthe cabinet–timesvaryaccordingtothelab• TurnofftheUVlightandturnonthefluorescentlight• Turnontheblowertocleantheenvironmentasyoumove items in and out of the hood• Adjustthewindshield•Wipedowntheworkingsurfacewith10–30%bleach•Wipedownyourworkingsurfacewith70%ethanol• ‘Sprayin,sprayout’everyworkingitemgoingintooroutofthe hoodwith70%ethanol•Whenfinishedworking,reversethesestepstocleanup•Whatequipmentwillbeneeded?• Howcanequipmentbedecontaminated?

When using biohazardous substances in the lab, or transporting them between labs:

• Carryonlywhatyoucanholdcomfortably• Closetubesandbottletopssecurely• Carryitemsuprightandlevel• Usegloves• Useasecondarycontainer• Useacartforlongerdistances•Movenewsubstancesin their original packaging

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Material Optimal Acceptable Do not useGenomic DNA -200C or less -200C - 40C for a few months > 40C

Plasmid DNA -200C or less -200C - 40C for a few months > 40C

Cultured Eukaryotic Cells, growing Growth temp to 40CWill stop growing at lower temperatures

Significantly above growth temp or freezing will kill them

Cultered Eukaryotic Cells, fixed 40C for years Room temp for several months Can be damaged by excessive heat

Eukaryotic Cells in storage media Liquid nitrogen NA NA

Whole tissue/Body Parts Up to -200C -200C - 40C > 40C

Bacterial cultures, growing Growth temp to 40CWill stop growing at lower temperatures

Significantly above growth temp or freezing will kill them

Bacterial cultures, pelleted -200C or less 40C for 1 hr or so > 40C

RNA -800C or less -800C to 40C > 40C for >18hrs

Whole blood -800C or less 40C for 1 hr or so > 40C

Saliva -800C or less 40C for 1 hr or so > 40C

Sampled cells (e.g. cheek swab) -800C or less 40C for 1 hr or so > 40C

Enzymes -200C or less Some ok at 40C > -200C

PCR Products -200C or less -200C to 40C > 40C for > a few min

Dinucleotide triphosphates (dNTP’s) -200C, can be thawed for regular use -200C to 300CMultiple freeze - thaw cycles. or prolonged exposure to heat can be damaging

Chemically Competent E.coli -800C, can be refrozen but not recommended Ice, 30 minutes > 00C for > 30-60 minutes

Storage

Toensureadequatecontainmentwhilepreservingtheiractivityorintegrity, biological materials must be stored properly.

Maintenance

Closeattentionmustbegiventotheroutinemaintenanceofthefacility, air handling systems and equipment that contributes to the containment of biohazardous materials.

Source:http://bitesizebio.com/9047/a-beginners-guide-to-storing-biological-materials/24 25

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Checklistforgoodbiohazardcontrol*Exceptional Laboratories will:•Designandrunprocessestokeepthespreadofcontaminants as low as possible•Considerallroutesofexposure•Selectcontrolmeasuresaccordingtotheamountofsubstance, how it gets into the body and how much harm it will cause•Ensureallmeasuresareeffective,easytouse,andworkproperly• IssueappropriatePersonalProtectiveEquipment(PPE)•Checkregularlythatmeasurescontinuetowork,andkeep records• Informworkersaboutthedangersandhowtousecontrol measures properly•Avoidincreasingtheoverallhealthandsafetyriskswhen making changes•Engageemployeesinsafety,healthandprotectionprograms•Employexpertpartnersinsafety,healthandprotectioncontrols

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