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BiologyandEnvironmentalScience~SafetyManual~MarylandPublicSchools
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INSTRUCTION&ASSESSMENT

DATAANALYSIS

SCHOOLIMPROVEMENT

IX.BiologyandEnvironmentalScience
RecognizingandControllingHazards
PrintVersion(135KBAcrobat)
BiologyandEnvironmentalScienceteachersandtheir
studentsfaceawiderangeofpotentialhazards.In
additiontochemicalreagents,therearethehazards
associatedwiththehandlingoforganisms,classroom
activitiesontheschoolgroundsandoutdoorstudyareas,
andthecontainmentofbiologicalspecimens.Effective
controlofsuchhazardsinvolvesboththerecognitionof
eachhazardandthedevelopmentofcontrolprocedures.

A.PersonalSafety
Everystudentandteachershouldbeprotectedbysafety
deviceswhenexperimentsarebeingconductedinthe
biologylaboratory.

BIOLOGYLABORATORIESARE
SAFEWHEN...
studentsafetyisconsideredin
determininganactivitysvalue.
properlaboratorytechniquesaretaught
andpracticed.
physiologicalmeasurementsareneither
stressfulnorinvasive.
careistakeninselectingandusing
reagents.
specimensarehandledaccordingto
professionalguidelines.

SeeChapterIV,PersonalSafetyProvisions.
1. BodyandClothingProtection
SeeChapterIV.C,ProtectiveApparel.
2. EyeProtection
SeeChapterIII.D,SafetyEquipmentChapterIV.A.,EyeProtectionConcernsChapterVI.J,
CryogenicsandChapterVIII.B.2.a,AttheSiteGoggles.
3. HandProtection
Rubberorplasticglovesshouldbeprovidedforstudentstousewhenhandlingpreserved
organisms.
Anyonewithbreaksintheskinorunhealedscratchesshouldwearrubberorplasticgloveswhen
workingwithpreservedorganisms.
Heavyrubberorleatherglovesshouldbeprovidedforusewhenhandlingliveanimalsas
protectionagainstanimalbitesandscratches.
Soapandwatershouldbeavailableforstudentuse.Studentsshouldberequiredtowashtheir
handsbeforeandafterlaboratoryexperimentsthatinvolvethehandlingofliveordead
organisms.
SeeChapterIV.C.2,ProtectiveApparelGloves.

4. RespiratoryProtection
SeeChapterIII.E,Ventilation,andChapterIV.C.4,RespiratoryProtection.

B.Classroom/LaboratorySafety
1. Equipment
SeeChapterVI,SafeHandlingofEquipment.
2. ChemicalReagents
a.Stains
Stainingisanimportantpartofbiologystudies.TeachersmustobtaintheappropriateMaterial
SafetyDataSheets(MSDSs)forallstainingreagentsandfollowthestatedprecautions.

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SeeAppendixC,MSDS:ExplanationandSamples.
b. DrugRelatedItems
Thefollowingsubstancescommonlyusedinthebiologyprogramhavespecialsecurityneeds
becauseoftheirpotentialabuse.Theyshouldbekeptinasecureareaandusedwithcaution:
Acetaldehyde
Adrenalin
Colchicine
Caffeine
EthylAlcohol(grain)

Histamine
Nicotine
Testosterone
Thiourea
Tobacco

SeeChapterVII.Chemicals:Managing,HandlingandDisposing.
3. Dissections

a.Studentsshouldwearchemicalsplashsafetygogglesandaprons.

b. Longhair,looseclothing,andjewelryshouldbesecured.

c. Rubberorplasticglovesshouldbeprovided.

d. Studentswithbreaksintheskinshouldwearglovesorbeexcusedfromlaboratory.
e.Theroomshouldbeadequatelyventilatedduringdissections,particularlywhenpreserved
specimensareused.

f. Careshouldbeusedinthehandlingofalldissectioninstruments.

g. Dissectingpansortraysshouldbeprovided.

h. Scalpelsorsingleedgedrazorbladesshouldbeused.Singleedgerazorbladeswitharigid,
reinforcedbackarepreferred.

i.Studentsshouldbeinstructedtocutawayfromthebodyandtocutdownagainstthe
dissectingpanortray.Caremustbetakentokeepthehandthatisnotholdingthecutting
instrumentawayfromthecuttingedge.

j. Itisimportanttosecurethespecimeninortothepanortray.Dissectionpansmaybeused.
SeeChapterIX.D.2.d,Zoology:AnimalConsiderationsDissection.

4. HeatingandSterilizationDevices

a.Autoclaves/DryHeatSterilizers/PressureCookers.Autoclavesanddryheatsterilizers
arepreferred.Allprecautionsindealingwithelectricalequipmentshouldbefollowed.A
pressurecookermaybeusedasasubstitute,butitinvolvesgreaterattentionduetothe
hazardsinvolvedinanonautomatedsystem.
Note:Mostplasticcontainersandequipment,suchasplasticpetridishes,arenot
autoclavable.
Autoclaves,heatsterilizers,andpressurecookersshouldberunonlybytheteacheror
professionalaideorbythestudentaideifhe/sheisunderthedirectsupervisionofthe
teacherorprofessionalaide.Theteacher,professionalaide,andstudentaideshouldbe
knowledgeableabouttheoperatinginstructionsofthepressurecooker,autoclave,orheat
sterilizer.
Inusingapressurecooker,checkthesafetyvalvebeforepressureisbuiltup.Finalgauge
pressuremustnotexceed15poundspersquareinch.Theequipmentshouldbeturnedoff
andallowedtocoolbeforethestopcockisopenedtoequalizepressure.Drythepressure
cookerbeforestoringbecausealuminumwilloxidizeifstoredwet,andpitsproducedfromthe
oxidationmayweakenthemetalwhenunderhighpressure.Heatsterilizersarepreferredover
pressurecookers.
SeeChapterXI.B,ElectricalHazards,andChapterIX.C.4,DecontaminationandDisposalof
Materials.
b. Gasburners.Whenheatingmaterialsathightemperatures,agasburnermaybeused.

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SeeChapterVI.D.1,SafetyRulesforUsingGasBurners.
c. Hotplates.
SeeChapterVI.D.2,SafetyRulesforUsingElectricHotPlates.
d. Waterbaths.Waterbathscanbeusefulinabiologylaboratory.Thebathsmustbewell
maintainedforsafeoperation.
e.Incubators.Incubatorsmaybeusefulcomponentsofabiologylaboratory.Careshouldbe
takentokeepincubatorssafeandwellmaintained.Theyshouldbecleanedoutregularlyto
preventunwantedgrowthoforganisms.Studentsshouldbeinstructedontheirproperuse.
f. MicrowaveOvens.Microwaveovensmaybeusefulinthebiologylaboratory,especiallyin
biotechnologyexperimentswhereagarmustbeheatedtopourgels.Microwaveovensshould
besafelylocatedandappropriatelymaintained.Studentsshouldbeinstructedontheirproper
use.Studentswithpacemakersshouldnotworkintheproximityofamicrowaveoven.
5. MicroscopeWork
a.Microscopes
Studentsshouldbeinstructedintheproperuseofthemicroscope.
Allprecautionsindealingwithelectricalequipmentshouldbefollowed.
Ifmicroscopeswithreflectingmirrorsareusedforillumination,caremustbetakento
preventusingdirectsunlightastheilluminationsource.
Studentswitheyeinfectionsshouldnotbeallowedtocontaminatetheeyepiece(s)ofthe
microscope.
b. Microtomes
Amicrotomemaybeausefultool,especiallyinupperlevelbiologyclasses.Studentsmustbe
instructedintheproperuseofthemicrotomes,whichcontainextremelysharpknifeblades.
SeeChapterXI.B,ElectricalHazards.
6. RefrigeratorsandFreezers
SeeChapterVI.E,RefrigeratorsandFreezers.

C.Microbiology
Thissectionpertainsprimarilytotheuseofviruses,bacteria,andothermicroscopicorganisms.
Bloodbornepathogensrequirespecialconsiderations.Thehandlingofthesepathogensistreatedin
29CFR1910.1030.Thispublicationcoversdefinitions,exposurecontrol,specificproceduresand
protocolstocomplywiththeregulations,precautionsforspecificpathogens,signs,labels,training,
andrecordkeeping.Properlaboratorytechniqueisthebasisforallcautionsinthissection.
SeeChapterIX.D,Zoology:AnimalConsiderations,andF,BiotechnologyandRecombinantDNA
Research.
1. MaterialsandSpecimens

a.Knownpathogensshouldneverbeusedintheclassroom.

b. Specimensshouldbeobtainedfromreliablesupplycompaniesorothersourcesthatcan
validatespeciesorstrains.Mostsupplycompanycatalogsindicatewhichorganismsare
pathogensandwhicharenot.

i.AlistofsourcesisavailableintheNationalScienceTeachersAssociationannual
publication,NSTAScienceEducationSuppliers.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nsta.org/scisuppNationalScienceTeachersAssociationscience
educationsuppliers
ii.Othersourcesmayincludelocalresearchfacilities,e.g.,universitiesorhospitals.The
AmericanTypeCultureCollection,anonprofitorganization,maintainsanextensive
collectionofmicrobiologicalspecimensthatmaybepurchased.Thissourceisespecially
usefulforobtainingmaterialsnotavailablethroughothersuppliers.
Thefollowingwebsiteoffersmoreinformationonthistopic:

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http://www.atcc.orgAmericanTypeCultureCollectionbacteria
iii.Specimensshouldberequestedforshipmentwhenneededandnotstoredforlong
periodsoftime.

c. Allmicroorganismsshouldbehandledasiftheywerepathogens.

d. Properaseptictechniquesshouldbeusedatalltimeswhenworkingwithbacterial,viral,or
microbialcultures.

e.Microorganismscultureddirectlyfromtheenvironmentshouldnotbeincubatedforcell
culturesasincubationcouldpromotethegrowthofpathogenicorganisms.

f. Humansand/orhumanproductsshouldnotbeusedasasourceofbacterial/microbialculture
materialinmostcases.Inspecialcircumstances(e.g.,APBiology),teachersshouldobtainthe
supervisorspermissionandfollowtheUniversalPrecautions.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://wonder.cdc.gov/wonder/prevguid/p0000255/entire.htmCenterforDiseaseControl
UniversalPrecautions
g. Bloodagar,serumagar,and/orchocolateagarshouldbeavoidedforuseinclassroom
experimentation.

h. Stainingreagentsmaybepurchasedthroughsupplycompanies.
SeeChapterIX.B.2.a,Stains,andIX.F.2,StainingDNA.

2. Equipment
Essentialequipmentforworkingwithmicroorganismsincludes:

a.Sterilizationequipment(autoclave,heatsterilizer,orpressurecooker)formediapreparation,
sterilizationofglasswareandequipment,anddecontaminationofdisposablematerial

b. Steriletransferequipment(micropipetterswithdisposabletipsorsterilepipets)forsafe
transferofmicroorganisms

c. Adequateworkspaceandequipmenttopreparemedia

d. Properstoragefacilities,includingrefrigerationandincubationequipment

e.Suppliesforcleaningupanddisinfectingworkareas

f. Pipets
Duetothenatureofmicroorganisms,theuseofdisposablepipets,pipettips,dishesand
cultureplates,etc.isrecommended.Ifyouusenondisposableglassware,takecareto
properlydecontaminateit.
SeeChapterVI.A,Glassware.

g. Specialtrashcontainersforallculturesforpropersterilizationanddisposal
Thereshouldbeseparatecontainersfordisposalofglass,plastic,paper,etc.Alltrash
receptaclesshouldbeclearlyidentified.
h. Petridishesforusewithnoninfectiousmaterials
UsesterileplasticPetridishesonlyonce.Afterusingthem,tapethedishesshut,place
theminabag,anddisposeoftheminanincineratororthetrashaccordingto
recommendeddisposalguidelines.

Glasspetridishesshouldbesterilizedbeforeuse.Afteruse,disposeoftheculturemedium
inasealedcontainerandsoakthedishesinstrongdisinfectant.Washthemindetergent
andautoclave.
SeeChapterIX.C.4,DecontaminationandDisposalofMaterials.

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3. ProceduresandSterileTechniques

a.Keepthelaboratoryclean.

b. Disinfecttheworkareabeforeandaftereachlaboratoryprocedure.Useofacommercial
disinfectanttowipedowntheareaisacceptable.

c. Studentsshouldusegloves,chemicalsplashsafetygoggles,andapronsastheteacherdeems
necessary.

d. Donotleavelaboratorymaterialsunattended.

e.Inoculatingloops

i.Inoculatingloopsshouldbeflamedbeforeandafterthetransferofmicroorganismsand
afinaltimebeforestoring.

ii.Theinoculatingloopshouldbeusedwithasteadyhandandshouldnotbeusedfor
stirring,assplashingmayoccur.

iii.Whentransferringtheinoculant,donotuseahotloop,whichcancausespatteringof
theculturemediumandtherebycreateaerosolsofthecultureorganisms.(Makesure
theloopiscoolbytouchingthelooptoanareaofthesterileagarthatwillnotbeused
orallowingthelooptoaircoolforafewseconds.)

f. Whenaliquidculturemediumisused,theliquidshouldneverbeallowedtocomeincontact
withthestopperusedtosealtheculturemedium.Careshouldbetakentoavoidspattering
whenthestopperisremoved.

g. Forcepsshouldbeusedtohandleslides,andslidesshouldbeflamedwithcaretoavoidburns
andshatteredslides.

h. Culturesshouldbeincubatedattemperaturesnohigherthan25Ctodecreasethepossible
growthofpathogens.

i.Pipettingofbacteria

i.Useamicropipetordisposablepipetandapipetterorpipetbulb.Micropipettersare
preferredfortransfersofsmallamountsofliquidinoculant.Adisposablepipetis
preferredforlargeramounts.

ii.Usedglasspipetsshouldbeimmersedindisinfectantandthenautoclaved.Donotallow
studentstoaspirateortospraybacterial/microbialcultures,whichcancreateaserious
biologicalhazard.Neverallowmouthpipetting.

iii.ColoniesshouldbecountedonclosedPetridishesorplates.Parafilmisrecommended
forsealingcultureplates.

iv.Anydemonstrationplatespassedaroundtheclassmustbesealedwithparafilmor
tape.

v.Anautoclave,heatsterilizer,orpressurecookershouldbeavailable.
SeeChapterIX.B.4.a,HeatingandSterilizationDevicesAutoclaves/DryHeat
Sterilizers/PressureCookers.SeealsoChapterVI.G,Pipets,andChapterVI.I,
Centrifuges.

4. DecontaminationandDisposalofMaterials
Disposalofscientificmaterialssuchasreagentsisanissueineveryscienceclassroomand
laboratory.Inbiology,itisnecessarytodifferentiatebetweeninfectiousandnoninfectious
materials.
a.Noninfectiousmaterialsincludematerialssuchaschemicalreagents,householdsubstances,
andbiologicalsamplesfreeofparasitesorcontagiouspathogens.Thesearesubstancesthat
carrynocommunicablehazard.
Noninfectiousbiologicalwasteshouldbetreatedwithsoundsafetymanagementtechniques.

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Todisposeofthesematerials,placethemindoubledomesticplastictrashbagssecuredby
metalwiretwists.Thebaggedwastesmaythenbeplacedindomestictrashreceptaclestobe
disposedofinanapprovedlandfillinaccordancewithstateandlocalregulations.
SeeChapterVII.C.3,DisposingofWaste.
b. Infectiousmaterials(orbiologicalwasteorbiohazardouswaste)arecommunicablebiological
materials.Thesematerialsincludecontagiousmicroorganismsorpartsofmicroorganisms
(includingbacteria,viruses,orDNAfragments)aswellasdisposablebiologicalequipment
thathasbeenexposedtoinfectiousmaterials.
Infectiousbiologicalmaterialsrequiredecontaminationpriortodisposalthroughoneofthe
followingmethods.
i.Incinerationonsite.Thisapproachisthepreferredprocess.Thismethodrendersthe
wastenoninfectiousand,atthesametime,changestheshapeandformofthewaste.
Schoolsthathaveincineratorsmustcomplywithallapplicableenvironmental
regulationsregardingairqualityandairemissions.Thenextmethodisemphasized
herebecausemostschoolsdonothaveonsiteincineration.
ii.Decontamination.Infectiousmaterialsmayberenderednoninfectiousby
decontamination(sterilization)priortodisposal.Belowarethemethodsof
decontaminationmostcommonlypracticedinhighschools.

Steamsterilizationinanautoclaveatapressureofapproximately15psianda
temperatureof121C(250F)foratleast15minuteswilldestroymicrobiallife,
includinghighnumbersofmicrobialspores.

Dryheatsterilizationmaybeused.However,thismethodrequirestemperaturesof
160170C(320338F)for24hours.
Note:Inbothcases,theautoclavesshouldbecalibratedfortemperatureand
pressureandmonitoredwithabiologicalindicator,suchasBacillus
stearothermophilusspores,toensureeffectivenessofthesterilization.Itisimportant
thatthesteamandheatcontactthebiologicalagent.Therefore,bottlescontaininga
liquidmaterialshouldhaveloosenedcapsorcottonplugcapstoallowforsteamand
heatexchangewithinthebottle.Biohazardbagscontainingwasteshouldbetied
loosely.Itisrecommendedthatbagsofbiohazardwastebeaffixedwithautoclave
indicatortapetoensurethetemperaturereadingsareaccurate.Oncedisinfected,
wastescanbetreatedasnoninfectiouswaste,doublebaggedindomesticplastic
trash/garbagebagsandsecuredbymetalwirecontainingtwistties.Treatedbags
andcontainersmaythenbedisposedofinanapprovedlandfillinaccordancewith
stateandlocalregulations.
SeeChapterVI.M,Sharps.
Ifneitherofthesesterilizationtechniquesispractical,theinfectiouswastemaybe
transportedoffsitetoaqualifiedmedicalwastedisposalfirmforsubsequent
treatmentanddisposal.
Ifnoneoftheaboveproceduresispossible,theinfectiouswastemaybeimmersedin
householdbleachfor610hours.Althoughchemicaldisinfectionisnotconsidered
completelyeffective,bleachisconsideredeffectiveinwipingdownexposedsurfaces
andequipment.

D.Zoology:AnimalConsiderations
1. Human
Noninvasive,nonstresslaboratoryactivitiesinvolvingstudentsasexperimentalsubjectsare
encouraged.Theseincludephysiologicalmeasurementssuchaspulse,bloodpressure,heartrate,
breathingrate,hearing,sight,etc.Obviously,everyprecautionmustbetakentoensurestudent
safety.
Thefollowingareexamplesofsafetyprecautionsthatshouldbefollowed.
a.BloodPressure.Whenusingthesphygmomanometertotakebloodpressure,donot
pressurizethecuffhigherthan150mmHg.Allowtwominutestopassbeforereinflatingthe
cuffwhentakingrepeatbloodpressuremeasurementsonthesameindividual.This
experimentcancausestressleadingtoashockreactionandunconsciousness.

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b. RespiratoryExperiments.Whenrespiratoryexperimentsaredone,rememberthat
hyperventilationcanbedangeroustoanyonebutparticularlytoasthmatics,epileptics,and
thosewhosufferfrombronchialconditions.Whenthespirometerisused,acleanmouthpiece
shouldbeusedbyeverypersonbeingtested.Whentestingforcarbondioxide,caremustbe
takennottoallowthetestsolution(bromothymolblueandcalciumhydroxide)toreachthe
mouth.Thisexperimentcancausestressleadingtoashockreactionandunconsciousness.
c. StethoscopeUse.Disinfectstethoscopeearpiecesaftereachuse.Teachstudentstheproper
useofthestethoscopetoavoidpotentialeardamage.
d. BodyFluidsandBloodbornePathogens
i.Recommendations.FederalRegulation29CFR1910.1030mustbefollowed.The
NationalAssociationofBiologyTeachers(NABT)makesthefollowingrecommendations
regardingtheuseofhumanbodysamplesinthebiologyclassroomorlaboratory:
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nabt.orgNationalAssociationofBiologyTeachers
Usesaferalternativestohumansampleswhenpossible.Materialsthatmimicthe
propertiesofblood,saliva,andurinemaybepurchased.TheAmericanBiology
Teacherjournalisasourceofinformationonalternatives.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nabt.org/journal.htmlNationalAssociationofBiologyTeachers
AmericanBiologyTeacher
Usesamplesknowntobefreeofdisease.Donotuseanysamplesofunknownorigin.
Avoidsamplesthatarenotcollectedatschool.
Usesamplesonlyifallpersonsincontactwiththesamplescomplywithallapplicable
UniversalPrecautions.Additionalguidelinesareavailablefromclinicallibraries,
hospitals,andpublichealthagencies.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://wonder.cdc.gov/wonder/prevguid/p0000255/entire.htmCenterforDisease
ControlUniversalPrecautions
InadditiontotheUniversalPrecautions,teachersusinghumanbodysamplesshould
conformtothefollowingprecautions:
Studentsmustbeallowedtocollectsamplesonlywiththesupervisionandadvice
oftheteacher.
Samplesmustbehandledwithplasticorlatexgloves,chemicalsplashsafety
goggles,andalaboratorycoatorapron.
Studentsmustalwayswashtheirhandsafteranylaboratoryactivityinvolving
humanbodysamples.
ii.BasicPrecautions.Belowisalistofbodyfluidsandwastesandtheirpossibleusesin
ahighschoolclassroomorlaboratory.Teachersmustfollowschoolsystempolicy
regardingparentalpermissionandstudentsrightstorefusetoparticipatein
experimentsusingbodyfluids.Teachersareencouragedtousesaferalternativesin
placeofhumansampleswhenpossible.
SomeofthematerialsonthelisthavebeenidentifiedbytheCentersforDisease
Control(CDC)asrequiringtheapplicationoftheUniversalPrecautions.Allbodyfluids
andwasteswarranttheapplicationoftheUniversalPrecautions.Recommended
precautionsandprocedures:
Treatallbodyfluidsandwastesasinfectiousbecauseitisnotpossibleinthehigh
schoollaboratorytodistinguishthatwhichisandisnotinfectious.
Takingbodyfluidsorwastesforexperimentsmustbeonavoluntarybasis.
Usesteriletechniquestopreventspreadinginfectiousdiseases.
SeeChapterIX.C.4,DecontaminationandDisposalofMaterials.
a.Bloodmaybeusedforbloodtypingstudiesandmicroscopicandphysiological
analysis.Studentsshouldbeencouragedtoobtaintheirbloodtypefromadoctor
andtoknowtheirbloodtypeincaseofanemergency.

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b. CheekCellsmaybeusedformicroscopicanalysisandcellcomparisonstudies.
c. Fecesarerarelyusedinbiologyinstructionbutmaterialscontainingfecesare
sometimesusedinmicrobiologyandparasitologystudies.
d. RespiratoryMucusmaybeusedinstudiesoftherespiratorysystem.
e.Salivaenzymesmaybeusedingeneralbiologyclassactivities.Cellscollected
fromsalivaareusedformicroscopicanalysisandforobtaining(culturing)oral
microorganismsfromtoothtarter.
f. Semensamplesmaybeusedformicroscopicanalysis.
g. Urinemaybeusedforurinalysisorcellculture,whichrequiresthecollectionof
freshurine.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://wonder.cdc.gov/wonder/prevguid/p0000255/entire.htmCenterforDisease
ControlUniversalPrecautions
2. Nonhuman
TheMarylandScienceSafetyProjectCommitteeendorsestheNationalScienceTeachers
AssociationsGuidelinesforResponsibleUseofAnimalsintheClassroomandtheInstituteof
LaboratoryAnimalResourcesprinciplesregardingthehumanestudyofanimalsinprecollege
education.Inallcases,teachersshouldconsultsuchorganizationsandagenciesasthelocal
HumaneSocietyandtheStateDepartmentofNaturalResourcesbeforebringinganimalsintothe
classroom.
Thefollowingwebsitesoffermoreinformationonthistopic:
http://www.nsta.org/handbook/animals.htmNationalScienceTeachersAssociationGuidelines
forResponsibleUseofAnimalsintheClassroom
http://www.hsus.org/programs/research/animals_education.htmlHumaneSocietyoftheUnited
Statesanimalcare
http://www.dnr.state.md.us/index.htmlDepartmentofNaturalResourcesanimalcare
a.Invertebrates
Invertebrateanimalsareoftenusedforobservationandlearningactivities.Forexample,
Drosophilasp.(thefruitfly),isusedingenetics.Anesthetizetheorganismscarefullybyone
ofthefollowingmethods.
i.Ifexperimentsaredonewithfruitflies,takecareinquietingthemand/orkillingthem.
Usingetherinkillingjarsisnotrecommended.Ifetherisused,itshouldbediscarded
withinamonthofopening.Onecommercialsubstanceusedasasubstituteforether
containstriethylamine(C2H5)3N,whichisflammable,toxicbyingestion,andasevere
irritant.Usewithcare.Othermethodsare:placingthefruitfliesinaPetridish,gently
coveringthemwithcotton,andtheninvertingthedishforexaminationunderthe
dissectingmicroscopeandrefrigeratingculturejarsandplacingchilledfliesonaPetri
dishoverice.
ii.Anesthetizingkitsalsomaybeused.Forexample,FlyNapkitscontainingrelatively
harmlesscomponentsmaybepurchasedfrombiologicalsupplycompanies.Any
anestheticshouldbeusedinaproperlyventilatedroomaccordingtothesupplier.
Teachersshouldobtainmanualsavailablefrombiologicalsuppliers.Thesemanualsare
inexpensiveandserveasacompleteguidetomaintainingandstudyingtheorganisms
intheclassroom.
b. Vertebrates(Nonhuman)
i.Donottakevertebratesfromthenaturalenvironment.Mostmunicipalities
prohibittheremovalofvertebratesfromthenaturalenvironment.Doingsoupsets
naturesbalanceandmayintroduceunwantedmicroorganismsordiseasedanimalsinto
theclassroom.
ii.Obtainanimalsfromacertifieddiseasefreesource.
iii.Whenstudyingdevelopingchickenembryos,donotuseanyembryosthataremore
than18daysold.
iv.Donotworkwithvirusinfectedeggs.
v.Disposeofdeadembryos,whichmaycarrypathogenicbacteria.

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SeeChapterIX.C.4,DecontaminationandDisposalofMaterials.
vi.Donotgiveawayorsellanyanimals,includingbabychicks.
vii.Donotreleaseanimalsthatarenotindigenoustotheareaintotheenvironment.
ReleaseofindigenousanimalsmustbeapprovedbytheStateDepartmentofNatural
Resources.
viii.MarylandStateLawprohibitsexceptforcommercialbreedingorraisinganyperson
fromsellingorgivingawaybabychickens,ducklings,orotherfowlunderthreeweeks
ofage.Thelawalsoprohibitsstainingorinanywaycoloringsuchananimal.Any
personwhoviolatesthislawissubjecttoa$25fine.
AnnotatedCodeofMarylandCommercialLaw,11904.SaleandColoringofChicks.
c. OtherGuidelinesforWorkingwithAnimals.TheMarylandScienceSafetyProject
Committeesupportsthefollowingadditionalguidelinesforworkingwithanimalsinwaysthat
protectstudentsandtheanimals.Mostoftheseguidelinesrelatetoanimalsintheclassroom.
i.Takecaretoavoidcontactbetweenhumansandanimalswheneitherofthemmaybea
diseasecarrier.
ii.Keeplaboratoryanimalsisolatedfromwildanimals.
iii.Onlythestudentassignedresponsibilityforanimalcareshouldhavedirectcontactwith
theanimals.
iv.Maintainagoodenvironmentfortheanimals,withamplefoodandwateravailableto
thematalltimes,includingweekendsandholidays.Keepcagescleanofwastes.
v.Protectanimalsduringtimesofpesticideuse.
vi.Sterilizecagesandequipmentbeforeandafteruse.Usehouseholdbleach,2%phenol,
orLysol.Rinsecagewellwithwater.
vii.Parentalpermissionmustbeobtainedbeforeallowingastudenttotakeananimal
home.
viii.Thefollowinganimalsshouldnotbekeptinschool:
venomousreptilesandfish
blackwidowandbrownreclusespiders
scorpions
bees,wasps,hornets,andotherstinginginsects
animalsathighriskofcarryingrabies
wildanimalsparticularlymammals
ix.Thefollowinganimalsmaybekeptatschoolwiththenotedcautions:
turtlesandsnakes(possibleSalmonellainfection)
furbearinganimals(possiblecauseofallergies)
tarantulas
parakeetsandparrots(possiblepsittacosisinfection)
x.Keepaquariumsandterrariumscleansothatorganicmaterialsdonotactasareservoir
formicroorganisms.Removemineralaccumulationswithavinegarsolutionandrinse.
d. Dissection.Bytheirconsistentattitudeinworkingwithanimals,teacherscanencourage
studentsunderstandingoftheeducationalvalueofdissectionwhilebeingsensitivetoliving
things.
i.Livingspecimensshouldbemaintainedinthelaboratoryuntilused.
ii.Liveanimalsbeingusedindissectionsshouldbepreparedusinganappropriatemethod
bytheteacherorbystudentaidesunderthedirectsupervisionoftheteacher.
iii.Specimenssuchasfrogscanbeheldforseveralweeksintherefrigerator.
iv.Preservedspecimenspurchasedthroughreputablebiologicalsupplycompaniesare

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acceptablesubjects.
v.Animalskilledonhighwaysandothernonpreservedspecimensshouldnotbeused.
vi.Certainspecimens,suchasfishandsquid,maybepurchasedfromthefrozenfoods
sectionofalocalgrocerystore.
vii.Teachersshouldassesstheirneedscarefullyandorderonlyenoughmaterialforayear.
Specimensshouldnotbestoredfromyeartoyearsincedeteriorationmayoccur.
viii.Beforeuse,specimensshouldbekeptintheiroriginalcontainersandplacedinanarea
notavailabletostudents.Decayedpreservedspecimensshouldbediscardedproperly.
ix.Preservedspecimensshouldbethoroughlyrinsedinrunningwaterbeforeuse.
x.Freezedriedspecimensthathavebeenrehydratedinadilute10%alcoholwater
solutionfor24hoursshouldbethoroughlyrinsedbeforeuse.
xi.Specimensarenormallypreservedinanalcoholbasedpreservativefromthebiological
supplycompany.Formalinorformaldehydeshouldnotbeused.Acceptable
preservativesusedbyreputablesupplycompaniesincludeCaroSafeandbioperm.
Specimensretainedforfurtherworkonsucceedingdaysshouldbelabeledand
refrigeratedifpossible.
xii.Animalskinscanbeprotectedfrominsectdamagebystoringtheminboraxor
mothballs.
xiii.Whileperformingdissections,studentsshouldwearchemicalsplashsafetygoggles.
Glovesandapronsshouldbeavailableforstudentuse.
xiv.Studentsshouldwashtheirhandsafteranydissectionactivity.
xv.Allequipmentusedindissectionsshouldbethoroughlycleanedaftereachlaboratory
session.
xvi.Studentsshouldbegivenadequatetimetocleantools,pans,anddissectingstations
beforetheendofthelaboratorysession.
xvii.Ifthedissectionistobecontinuedatalatertime,specimensshouldbeplacedinplastic
bagstopreventdessicationanddeterioration.Bagsshouldbeclearorclearlylabeled.
xviii.Specimensshouldbebaggedandthendiscardedinanappropriatemanner.
SeeChapterIX.C.4,MicrobiologyDecontaminationandDisposalofMaterials.
xix.Chemicalpreservativesshouldbediscardedaccordingtothedisposalinstructionsinthe
MSDSforthesubstance.Attheconclusionofthelaboratoryperiod,everyonemust
thoroughlywashhandsandarmswithsoapandwater,takingcaretocleanunderthe
fingernails.
SeeChapterVII.C.3,ChemicalWasteStrategiesDisposingofWaste.
e.ResearchProcedures
i.Useinvertebratesforresearchwhenappropriatebecauseoftheirvarietyandthelarge
numberofspecimens.
ii.Vertebratesareappropriateincaseswheretheirsimilaritiestohumansareimportantto
theresearch.Researchshouldbecarriedoutwithqualifiedadultsupervisionandthe
adviceofaveterinarian.
iii.Plantsshouldbeusedwhereverpossibleforexperimentsonorganisms.
SeeChapterIX.E,BotanyandMycology(Fungi).

E.BotanyandMycology(Fungi)
1. FacilitiesandEquipment
a.Facilitiesnecessaryincludeproperlighting,adequateheat,adequatewater,andadequate
nutrients.
b. Containersshouldbecleanedbeforeandafteruse.
c. Commercialpottingmixturesarerecommendedovergardensoilbecausetheyarerelatively
sterile.

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SeeChapterIX.G,GreenhouseMaintenanceandOperation.
2. Cautions
a.Allergies.Manypeopleareallergictopollen,moldspores,orotherplantexudates.When
usingflowers,mushrooms,fungi,etc.,inthelaboratory,adequateventilationisessential.
PollenandmoldsporesshouldbedisplayedinclosedglassPetridishes.
b. Seeds.Studentsshouldnevereatanyseedsusedinthelaboratory.Whenworkingwith
pesticidetreatedseeds,theseedsshouldfirstbewashed.Studentsshouldwashwithsoap
andwaterafterhandlingsuchseeds.
c. Thorns/Needles.Manyplantshavethornsorneedles.Thesemaybeveryannoyingoreven
dangerousifcontactismadewiththeskinoreyes.Studentsshouldbemadeawareofthe
dangersofhandlingsuchplants.
d. ToxicPlants.Certainplantsandplantparts(aswellasfungi)containharmfulsubstances.
Somearepoisonousuponskincontact(e.g.,poisonoakorpoisonivy).Gloveshelptoavoid
skincontact.Otherplantsarepoisonouswheningested(e.g.,foxglove).Noplantshouldbe
ingestedinthebiologylaboratory.Studentsshouldbemadeawareofpoisonousplantsand
beabletoidentifycommonpoisonousplants.LocalhealthdepartmentsortheMaryland
PoisonCenter(MPC)arevaluableresourcesforsuchinformation.PhonenumberfortheMPC:
18004922414.
e.Disposal.Exoticplantsshouldneverbereleasedintotheenvironmentwheretheymay
competewithlocalplants.Suchareleasecanresultinanimbalancetothenaturalflora(and
eventuallyfauna).Forexample,thekudzuplantandpurpleloosestrifehavebecomepestsof
majorproportions.
Nativeplantsnormallydonotpresentaproblemforthelocalenvironment.Suchplantsshould
bediscardedinamannerconsistentwithschoolpolicyandlocalordinances.
3. Chromatography
a.Chemicalsplashsafetygogglesandapronsshouldbeworn.
SeeChapterIV.A,EyeProtectionConcerns.
b. Onlywaterbathsorhotplateswithwaterbaths(andnotopenflamefires)shouldbeusedfor
chlorophyllextraction.Extractionmayalsobeaccomplishedbyleavingtheplantmaterialin
thesolventsovernightatroomtemperature.
c. OnlyPyrexorcomparableglasstubesshouldbeused.
d. Dissolvinganddevelopingsolventsgiveofftoxicvapors.Theymustbestoredinclosed
containersandtheroom
e.Solventsarehighlyflammableandmustnotbeusednearanopenflame.Avoidskincontact
whensprayingthedevelopingsolvents.Useafumehoodwhenappropriate.
SeeaboveChapterIX.D.1.d,Zoology:AnimalConsiderationsBodyFluidsandBloodborne
Pathogens.

F.BiotechnologyandRecombinantDNAResearch
Workwithdeoxyribonucleicacid(DNA)isatthecoreofmanyofthehandsonactivitiesinmolecular
biologyandbiotechnologythathavebeenintroducedintothehighschoolbiologylaboratory.Thestudy
ofthechemicalandphysicalpropertiesofDNAofteninvolvesthespooling,isolation,enzymatic
digestion,gelelectrophoresis,andmanipulationofbacterialcellstointroducenewgeneticinformation.
Manysuchlaboratoryactivitiescanbepurchasedascompletekitsthatprovidedocumentationand
guidelineshelpfultobothstudentsandteachers.Thesekitsareespeciallyrecommendedforteachers
whoarenotfamiliarwithstandardproceduresinresearchlaboratories.Safety,asalways,isacrucial
partofanymolecularbiologyexperience.Researchrequiringcontainmentisprohibited.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nih.gov/od/oba/NationalInstitutesofHealthNIHGuidelinesforResearchInvolving
RecombinanyDNAMolecules
AllresearchinvolvingrecombinantDNAtechnologymustbecarriedoutinaccordancewiththeNational
InstitutesofHealth(NIH)guidelinesforconductingresearchusingrecombinantmoleculesand
organisms.TheseguidelinesarecontainedintherevisedNIHGuidelinesforResearchInvolving
RecombinantDNAMoleculesdatedJune24,1994asamendedthroughApril30,1998.Essential
guidelinesforhandlinganymicroorganismorDNAmoleculeinthelaboratoryarealsocontainedinthe

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StandardMicrobiologicalPracticesectionofthemanual,BiosafetyinMicrobiologicalandBiomedical
Laboratories.Theguidelinesincludeproceduresforhandlingchemicalsandmicroorganisms,
maintainingasafeworkplace,anddisposal(includingdecontamination)ofusedmaterials(including
cells).Themanual,publishedbytheU.S.DepartmentofHealthandHumanServices,isavailableonline
fromNIHandCDC.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nih.gov/od/ors/ds/pubs/bmblNationalInstitutesofHealthBiosafetyinMicrobiological
andBiomedicalLaboratories
Escherichiacoli(E.coli)isthestandardexperimentalbacterium.E.coliisanormalresidentofthe
animal(includinghuman)digestivetract.ManystrainsofE.coliareknown.Afewstrainscancause
diseasesinhumans.StrainsofE.colirecommendedforlaboratoryuseareengineeredsotheycannot
normallysurviveoutsidetheprescribedconditionsofthelaboratory.Therefore,thesestrainsposelittle
riskofcausingdisease.However,anyopportunisticpathogencancause.problemsifappropriatesafety
precautionsarenottaken.Itisimportant,therefore,toadherestrictlytoacceptedmicrobiological
practiceswithallmicroorganisms.
1. Guidelines
Theguidelinesbelowsummarizetheproceduresforworkingwithbiotechnologytoensurethatthe
activitieswillbeperformedsafely.
a.HandleallmicroorganismsandDNAcarefully.Treatthemasiftheycouldcauseinfections.
b. Donoteat,drink,orapplycosmeticsinthelaboratory.Keepfingersandwritinginstruments
awayfromyourfaceandmouth.
c. Handsshouldbewashedwithsoapandwaterbeforeandafterhandlingmicroorganismsand
beforeleavingthelaboratoryregardlessofwhatmaterialswereused.Whenhandling
microorganismsorotherlivingmaterials,studentswhohavecutsontheirhandsshouldwear
latexorrubberglovestoprotectagainstinfection.
d. Useonlymechanicalpipettingdevicesfortransferringanymaterial.Donotallowmouth
pipetting.
e.Performprocedurescarefullytominimizetheformationofaerosols.Forexample,inclose
proximitytoliquidsurfacesorthebottomofemptyreceivingcontainers,pipettipstendto
formaerosols.Donotforcethelastdropfromapipet.Keeppipettipsawayfromthefaceto
avoidinhalinganyaerosolthatmaybeformed.
f. Decontaminateworksurfacesbeforeandaftertheiruseandafteraspill.Decontaminating
solutionsshouldbereadilyavailableandcontainedinwellidentifiedlaboratorysqueeze
bottles.
g. Discardinappropriatelymarkedcontainersallsolidandliquidmaterialsthathavecomein
contactwithmicroorganisms.Thecontainersshouldbeeasilyaccessedbystudentsateach
laboratorystation.
h. Decontaminateallliquidandsolidwastesthathavebeenincontactwithexperimental
microorganisms.Destroyexperimentalmicroorganismsbeforedisposal.
i.Glassware(includingpipettipsandEppendorftubes)thathasbeenincontactwithisolated
DNA,restrictionenzymes,orothernonlivingmaterialsdoesnothavetobedecontaminated.
Itshould,however,besoakedinadisinfectantsuchasahouseholdbleachsolutionforan
hourandthencleanedthoroughly.Glasswareandotherequipmentthathasbeenincontact
withharmlessmicroorganismsmaysimplybewashedthoroughly.
j. Wearingchemicalsplashsafetygogglesinthelaboratoryisrecommended.
SeeChapterIX.C.4,MicrobiologyDecontaminationandDisposalofMaterials.Guidancemay
alsobeavailablefromauniversityorresearchlaboratory.
2. StainingDNA
a.Methyleneblue(oracommercialderivative)istherecommendedstainingagentforviewing
DNAaftergelelectrophoresisinthehighschoollaboratory.
i.Studentsshouldwearlatexglovesinhandlingthisstainbecauseitismoderatelytoxic
andwillstainskin.
ii.Indisposingofthisstain,followlocalregulations.Donotpourmethylenebluedownthe
drainwithouttheapprovaloflocalauthorities.

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b. Ethidiumbromide,astainingagent,shouldonlybeusedby,orunderthesupervisionof,a
scientistinafacilitywherenostudentexposurewilloccur.Whileitismoresensitiveand
quickertousethanmethyleneblue,itisamutagenicagent.
3. ConductingGelElectrophoresis
a.Gelboxespurchasedfromabiologicalsupplycompanyarerecommended.Theseboxesare
safeforstudentuseifinstructionsarefollowed.Ifhomebuiltboxesmustbeused,they
shouldbecarefullyconstructedforsafety.
b. Electrophoresisgelsarerunathighenoughelectricalvoltages(75140volts)tocausesevere
jolts.Studentsmustbewarnedagainststickingfingersorelectricityconductingmaterialsinto
theelectrophoresisbuffersolutionwhilethegelboxisinoperation.
4. Radiation/Radioisotopes
RadiationexperimentsandtheuseofradioisotopesarehighlyregulatedbythestateofMaryland
andthefederalgovernment.Theteachermustbeawareofspecialprecautionsneededtoworkwith
nuclearmaterials,includingissuesrelatedtothenatureoftheradioactivesources,studentcontact,
securestorage,anddisposal.Therearealsolicenserequirementsforpersonswhopossessnuclear
materials.Fordifferentisotopes,federalandstateregulationssetdifferentlimitsforpossession
anduse.
SeeChapterXI.F.3,Radioisotopes.
Thefollowingwebsiteoffersmoreinformationonthistopic:
http://www.nrc.govNationalRegulatoryCommissionisotopes

G.GreenhouseMaintenanceandOperation
Forschoolsthathavegreenhousesavailableforbiologyandenvironmentalclasses,thefollowing
guidelinesareintendedtoaidintheirsmoothmaintenanceandoperation.Theseguidelines,which
supplementapplicableschoolregulations,applytoanyindividualworkinginthegreenhousearea,
studentorteacher.
1. Guidelines
Thefollowingguidelinesaredesignedtoensurethatallgreenhousecomponentsarefunctioningat
anadequatelevelforoptimumplantgrowthandatasafelevelforstudentuse.
a.Checkwaterlines,heatingsystem,fans,andtemperaturecontrol.Theseareusuallyroutine
proceduresthatcanbecheckedbytheschoolmaintenancestaff.
b. Makesureallautomaticequipmentisfunctionalandaccurate.
c. Cleantoolsafteruseandstorethemappropriately.
d. Instructstudentsintheproperuseof,andconductin,thegreenhousearea.Itis
recommendedthatstudentsberequiredtoobtaintheteacherspermissiontoenterthe
greenhouse.
e.Ruleswhichapplytothegreenhousemustbeclearlystatedandexplainedtostudents.Itis
importantthatstudentsunderstandthattherulesareforthesafetyofboththeorganismsin
thegreenhouseandthestudents.
f. Studentsandteachersshouldbecautionedtohandlefertilizercarefullytoavoidinhalingthe
dust.
g. Washfruitsandvegetablesbeforestudying.Eatingfruitsorvegetablesthathavebeen
cultivatedinthegreenhouseisnotrecommendedunlessspecialcarehasbeenmaintainedin
thegrowthofsuchplants.
h. Inspectthegreenhouseperiodicallytopreventthecultivationofunlawfulplantssuchas
marijuana.
i.Makesuretomaintainadequateventilation.Ventilationisespeciallyimportantwhenusing
pesticides.
SeeChapterIX.G.2,GreenhouseMaintenanceandOperationPesticides.
j. Useorganicmethodsofpestcontrolwhenpossible.
k.Maintainallequipmentsoasnottoimpedethesafemovementintoandaboutthe
greenhouse.Forexample,hoselinesshouldbeproperlymountedandstoredtokeepthefloor

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clear.
l.Washhandsthoroughlyafterworkinginthegreenhouse.
2. Pesticides
a.SelectingPesticides
i.Usetheleasttoxicpesticides.
Notesignalwordsfoundonpesticidelabels:
Danger=highlytoxic.
Warning=moderatelytoxic.
Caution=slightlytoxic.
Nocautionorwarning=relativelynontoxic.
ii.Thesafestinsecticidescontainpyrethrins.
b. UsingPesticides
i.Pesticidesaretoxicandshouldbeusedonlyaccordingtoinstructionsoncontainer
labels.
ii.Pesticidescanenterthebodythroughtheskin,mouthornose.Beforeusingpesticides,
coverupexposedskinwithwaterrepellentclothesandboots.
iii.Wearawidebrimmedhatandafullfaceshield.
iv.Useunlined,naturalrubbergauntletgloves.
v.Useexhausthoodsandventilationsystemswhenspraying.
vi.Donottouchthemouthorfacewithhands,forearmsorclothing.
vii.Donotexposeadrinkorfoodcontainertopesticides.
viii.Washhandsandfaceimmediatelyafterapplyingpesticides.

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