lab skills full lecture scanned

8/11/2019 Lab Skills Full Lecture Scanned

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Skiltrln BlologlcrlSclences-SB1203

Lecflre& Practical Session:

Basic& AdvancedLaboratorySkillsandInstrumentCalibration

Prcprredby: Dn Kuilrn Tennrkmn

Thelearningobjectiveof this ptacticalisto impnove andfollow

)nurabilityto understsnddirectionsina Biology labontoryclass

Oneof the most important skills for thoso entering is the

thefieldof BiologicalSeie.nces,ability to dospccificallywhataprntoeollexperirnent from this sanhave

requirc$.Deviatingdisastrnusconsequence$. I procedurcs

Instuctionsforexperiments arcusually wriffen vcrycarefullyandrcquire that aper$onbring complete and undishacted todrem.

attention

Thisability is importantnot only to successfully aprocedurr but

complete orexperiment,alsobecausein any ficld of sciences, calledupon to teach othert.

ons is fiequently

AsaBiologistothereisa demand thatapersonbe attentive and skilled enouglr to seeaprocedurcdemonstrated hir# hersol{, and in that doing,

once,then right awaydotheprocedurnbecomesoqualifiedthatthepersoncan then turn around andteach the nextp ffion.All of thisrequiressarefulattentionto insEuctions, andcarefi,rlattentiontowfratyoustudoing,

yourabilityto understandby asking youto do a few lab procedures

lnthis lab class,I will wor{c to improve and follow directions

independmtly.

Thesedirectionswill be both written, andoral. Sothelabenvironmentwill beonEin whichyouwill be challenged to improve skills. Your skills will also be sharpened

yourattention inthiswayby being ehallengedwithproblem-solving which also requirecarcful rcading and

questions,attentionto detail.


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At the end of the pruc'tical,youwill be able to do a self evaluation-howskilledyouareatcarefullyreading(orhearing),interprctingdirwtions, and puttingthem into action.


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t. HsndlingChemicnls/ LiquidsInpracticalclassesthe person in charge (thatis lesturer) hasaresponsibilityto informyouofanyhazardassociated andalso expensive instruments. as

withchemicals I delicate However,studentsoneof first duties when usingunfamiliarchemicals/ equipmentisto find out theproperties(ofchemicals)andoperatinginstructions(ofequipment).

Ualng ehamlcals rcsponelbly becanaidorate to othara: alwaYs returnstararaom chemicals promptly to thecorrect place,frapertwhen suppliesareganing lsw to the person who loaksafter storagefordaring,lf yau emp* anaspiratoror wash bottle,filt it up from thaappropriatesource.

Finding out about chemicals TheMercklndex (O'Neilot al,, 200c/ and theCRC Handbook of Chemistry and Physics

(Lide, 2006) are usaful sources of infor"matiort on the physicatand biotogicalpropertiesaf chemicals, inatuding meltingand boiling points,solubilifi, toxicity,etc.

'':

(.

tsFr. Sodium Chlorlde, [7647-14-.11Salti conrmon salt,ClNs: mol wt 58.44. Cl @.679{., N0 19.:14%.NaCl. Thc aniclerrf curnmgfce ir also kno\.vn as ubla salt, t'p.cksaL ar sca salt.

Occwr in ileruna qsthe minonrl Aolrrc, ltudusld by mining (rwk$s.lt),t,y er-fiFarstionof hrlne tiorn undurground salt deprosits ondfronr ncr rva[erby solcr *veporation: Faith, I(a"res& Clark't Lndfiltrid!Chc:mit:uis,F. R. Lowonhetrn, M. K. *tnrnn, E&. fltrifbylntrrclen


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*ol rn nlcohol, Its soly in rvrter ir decrca*cd by llcl. Almost inrolin concd H(1, Itr oq soln ir neutrirl, pH: 6.?-7.3. d of satd aq solnat 15' ir 1,202, A 2396 nq soln of sorliunr chlortde frecaes at-20.5"e ls-F). LD50 orllly in rtts: 3.75 *0.43 g/kg (Boyd,Shnnnc),

Nt.tte: Blasalt. s brnnd of sodhrm chloritls conl8 tracc amounts ofeohnlt,iodirre,iron, eopper, mangnncss, zinc is us d in fern anirrnh.

UsE: Ntrturiil ralt i.r tlrc vrunceof chlcrine nnd of sodiumaswellac of cll, or practicallyull. thcir {,'omf}d.r,c.9.. hydrocirloric acid,cldclrster,godiumcnrtxmutc. hydroxidc, ctc.: for prescrvingt'mids;mnnuf soep, to $altoul dyeu in frtezing mixtures: lbr dycing lndprintingt'ahrict,glaa,ingpott*ry,curiug hid*s: mctallurgy of nn andothcrmeBls.

TuIdRApcAt: Elactrolytn replenisheri enrctic;topical anti-in'fiammrtory,

TtmRAPfATlvtgrr: ljcnentialnutricntf&e tor. May bc givenorullyur srrrctic, slomachic. laxativc or to ctimulatcthirst (prevclrtionofcalculi), Intravcnou,clyaa isotonic solutionto rnise bl


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You should alwavntreat chemicals as pot ntiallydangerous, theeegenoralprecautionol

following

.Donot um any chrmlcal untll youhrveconlldrredtha rlrkr Involvod * for lab classeofyou shouldcarefullyread all hsrard and risk informstisnprovidedbeferayou stsrt work. In proiectwork,youmaynEed to be involvadin therisk.aeseasmentprosefifiwlthyour$upervitor.

rWaara hhoratoryaoatat alltlmoc * the coat thouldbefully faatencd andeleaned should

appropriately,

any ahemlcal eompoundbenBilledon it' Cloaod-toefnotwoar will protoctyourfeetshould eny spillagaeoccur.

rMaka ture youknowwhsre the rafstY apparrtua lrkept beforayou bcgln worklng this epparatus

-lncludeseyobath, fira eninguiehorsand blanket.first ald kit.. Wrar rafaty glarrar and glover whon worklngwlth toxle, lrrltrnt or corrotivoshamlcals.andforanyrubatanettwherc tha hamrds ate not yetfullyeharacterlaad make sure you underatandthehazardwarningaigna{p.120}alongwlthany speclfic

oystemsutprscedurae inafumecupboard,

hauard-codlng ueadinyourdepartment.Carry

wfthEolid motarialUrealdrrudr ar plpenaflllen to mlnlmln tha rl* ofcontrc'tudth ha-mdourroh*lonr -theseaids are

furthar dsraifcd onPP.122'3,Nevarlmoks, eat, drfnk or chaw gum In a labwhrre chcmlealsarahandlad-thiawill minlmieethorigkofingaetion.

L*ol 'afl solutionr approprtataly uso theappropriateharard warning information(seepp.120,1331.Rcportall rBlllageo of chamforhlrolutlonr-makesurethat Epillagea upproperly.

arEcleaned$torc harardsuEchemicalsonly tn the approprlatolocatlona-for examplera spark-prooffridgo isrequlredforflammableliquids;acldsandeolvents


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shouldnotbe stored together.Dlrporo of ehrmlcalrIn thr corractmannrr -ifunsur ,aeka memberef saff (donot agaumc thatit iaeafuto uaothelabwastebinortheainkfordiaposal).Wmh hrnda rftar any dlrcst osntaet wlth chcmlcalr

-

orblochcmlcalmatorial alwaVgwaehyourhandsattheend of alab session,

pipetteo hruette

(a)Youareprovidedwith a meshenieal glasspipette, and a conicalflask,explain measuring tomeasurefollowingvolumesforveryprecisepreparations solutions:

Givingreasons theappropriete devicethatcanbeused the

of standard500ml,50ml' 5 mlr lpl.

(b) Brieflystntetheproblemsthat one canfacewhenhandlingfollowingliquids.

SuggestONElogicalwaythatyoucan adopt toovercomesuchproblsms whenhandlingliquids.

eg.Highviscosityliquids:Problem-dfficult to clispenseSuggestion

to over cometheproblem-allowingtimefor all the liquid ta trans.fer.

(i) Handlingorganicsolventssuchas ethanol, acetonedifferentlycharged

(ii) Usingpolarand non polarsolventmixtureto separatecompounds(iii) Maintainingcellcultures/ suspensionsq


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(e) Teehniqueof making up an aqueous solutionof knowneoncenffationftomsolidmaterialand also preparationof asingle dilutisn using a stock solution,firgredfer.'

(i)List the procedurethat has to befollowed to prepare250 ml of 0,1 moll-lNaCl(noto:moleculnrmas$ofNaGlis 58.44)(ii)tist theprocedurethatyouhaveto followwhen l00ml of 0,2M NaClsolution hsE to be made using astoeksolutionof 4M NaCl solutionavailablein thelaboratory.(iii)Briefly explain theadvantageof preparinga decimal dilution serieswhenyouaregiventlre task to make a l0'' final dilute(10ml) from a fullstrengfih of 10m1.solution

(d) Fromthe list of terms: pnrtr per thoucand, eompositionpereent % wfty,ppm,andpercentcomposition9/o w/v, select alternative

themostappropriate term that can beusedtoexpresstherelativeamountsstrtedbelow:

(i)psml'r(ii)l0'69 ml(iii)pss'r(iv)mg g'r(v)solutemassin(g)per1009of solution(vi)solutemas$in(S)in l00ml of solution

+


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?. pHondBuffcr Solutions

l,tjl--,:-coynound thatactsa s

proton

donorIn'aqueous

solution.-

r compoundth t aetEaEa

lT:

protonacc ptor

Inaqueou,soluiioi.fSllueateptll -anacidtogetherwtth

rtBcorresponding

base,

Afkati-.a

compoundthat liberstesftvdroxytionswhdnit ciisd;itur.iin.,trydroxyllonsare *tr6nJiv'iujl,' ,rri,

wittraduce

the proton eoicbniraiilh.||plfUo * a cornpound'i thatcanact

1sbgth.anacidand b ;;,rctJ,i, ,ol|:fh:rvt* sincait ,iy eiiiilt-iltuto

srvea protonald g' htd;;iyi'' ion

(amphoteric

behavrour).

pHmcarurcment

pHisameasureof theamount.o{hrdlogen

ions(H+)ina solution: thisaffectsthesolubility

ofmanysubstances

andtheactivityofriosiuiorosiealsystems,fromindividualmoleculesto

wholeorganisrns.

It isusualtothinkof aqueoussolutionsascontainingH+ ions. (protons), though protons


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aetuallyexistin theirhydratedform,* rtyoroniumions(Hro*).

pHisexpressed

in negativepowers

of 10andit iscustomarytouseapH$eale.

pH = *lo916

lH'l

[H+]is the Brotonactivityin moll-'

pHvalue [H+l(rnoll-1)

B I x'io;P

7 1 x 1017,

I 1x104

mogn 3.?x 10-7-logto mean 6.43

AlwaysrememberthatpH scaleisa logarithmic one, not a linearonq a solution withapH

3.0isnottwiseasacidicas a solutionofpH6.0;butonethousandtimesacidie(i.e.coniains 1000timestheamountof H+ions).Therefore,youmayneed to convert pHvaluestoprotonconcentrations

beforeyoucaqyoutmathematical

manipulationsllBetheboxgivenq,b Sve

,


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rnfhat i$ tr';r*d*t & bas,c ?

The vulue rvhcre

an equalam{cuntof H* and oFl" ionsarepre$entistermedneutrolity:at.25"c lhepH ofpurewarerat neutri*lityis 7,0.At this

temperature.pH valueshelow?.0ure aciclic rvhile valucsahove7,0nre

alKiltlne.

Tahle. Propertiesof some pHindicatordyes

Acid-baaecolourDye change

Thymol hlue(aeid) red-yellow

Bromophenolbluo yellow*blua

Congored blue*rcd.

Bromocrasolgre9n yellow*blue

Reeazurin orenge*'violetMethylrod red-yellowLitmus red-blueBromocresol

purple yellow*purple

Bromothymolblue yellow-blue

Neutralred red-yellow

Phenol red yellow*red

Thymolblue(alksllne) yellow*blue

Fhenol"

phthalein none-rad

UsefulpHrange

1.?-6.8

1.2-6.8


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3,0-5,2

3.8-5.43,8"6,54.3-6,14.5*8.3

5:8-6,8

6,G-7,6

6.H.06,8-8"28,0-9.68.3-10.0

Measuring pH

pll electrodesAccurntep[{ nreanurements usingl couplerlto n

canbernnde * pHclectrode.pH mcter.ThepHcleetrode a cermhination comprising

isusually $lcctrocle.tw()sefurrate$yst$m$:un H+-sensitive glilsselestrodennd a referenceelectrodewhich is unaffected by l{f ionconcenrfltion(Fig,2' )' Whcnthisis immersedin er solutioR, voltitge hetrveen

a pH-dependent thetwoelectroelescanbenreasureelusing In nrost citses.

a potentiometer. thepllelcctrc.rcle (containingthe glassand ret'ercnce is

arssembly electrocles)connectedto a separate pH meterhy a cable. althouglr $CImehnndheltlinstrument$(

pll probes)havetireelestrodesnnd metsrwithintltesameassembly. field effect transistor of

afrenusinganH+-sensitive inplacea glasselectrode,to itnprovedurabilityandportaLrility.

-i

p'II indicafnrd.1'e'.1 *

Thesel'ompounds(usuallyweakacids)chartgecolnurin a pll-dependentmanner.They nay be added in small&mount$toa solutioll, or thcy cnnbeused in paperstripfbrrn.Eachindieatorelye usually changes coltturoveril

rcstrictedpH rflnge.typicallyl*2 pH units \ : uttivcrsalindieatordyesT.papers

makeuseof u combinationof individual dyestrtmea$urea wider pH rangc.Dyesare not suitable. for accuratepHmeasurement by other components of thc solution

asthey are affectedincluclingoxidizingand reducingagents attd salts.However.the-vare


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usetbl for:

. estimatingthc approximate pH of a solution;I cletermining in pH, for example nt the end-pointof it titration

;rehunge0rtheprclductionofacidsdrrringbacterierlmetaholisnri. estnblishingthe approximate pH of intnrcellularcompartments,for

'vital'stnin,; \

exnmpletheuse of neutral redasa

lfierilrurlng PH -the PH of a neutral ,solutionchangeswith ternparatura( *.

n

, ", dtteto the enhaneeddissaciationofwaterwith increasrngtemperatare,Thismust be taken into acoount whenmaasuringthe pH of any solution andwhenintarpretingyourresufts.

SAFETYNOTE Preparing a diluteacid solution uaing concentratedacid always slowly add the can

-centrated acid to watar, not therevarse,srncethe stranglyexothermicprocesscan trigger a violentreactianwith water,

$AFEIY NOTEPreparingan alkali

solution typicalty,tha alkallwill be-

in solidform (e.9.NaOHIand addi'tion to water will rapidlyraise the.temperatureaf the solution: useon ly heat-restsfanf ret coaled

g/asswawithwaterif necessary.


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Thefollowingproeedureshouldbe usedwhenever

!o:u,tnCIksa pH measuremsnt:consultthe manu'facturer'shandbookfor spscificinformation,wherenece$sanl/,

Donotbe tempted to miss out any of thestepsdetailedbelow,particularlythoserelatingtotheeffects of temperature,oryourmeasurements

arelikelyto bo ihaccurate.

porouspluu

H*-gen*itivgglesrolectrodeHCIsolution

solution

Fig, 2 Mea*urentent of pH using a cnmbinationpFletectrode and rnster. The electrical potentiel differeneereeord*d by the potentiomsteris direetlyproBortionalto theBHoftheteeteolirtion.

1, $tlr tha taot *ofution thoroughly befsre Youmakeany mae*urFmgryfiit is often bestto u$e Imegneticstirrer, Lepve the solution for suffisianttimstoallowEquilibration

atlsbtemBerfltur .

3. Racord tha tamparaturaof every aolution youuts,

includingall eslibrationetandardeand samples,sincothl*willaffectKn,,neutralityandpH,3, $s{ tha tamperntura compsnsator on tha rneterto ths aBproprlatevalue. This controlmakesan

allowance for the effeet of temporature on the',":electrical pot ntial difference iecorded by the tmeter:it doesnof allow for the othertemperatur -idependent effects mentioned elsewhsre. Basicrl'instrumentshave no temperaturecompensatlr,:..and should only be used at a epscified':temperature,either 20qCor ZE'C, otherwise th'ay ''

'

will not give an accurate measurement'More'

sophisticatLdsystemshaveautomatictemperature

compensation.I


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,4, Hin*a the electrode assombly with distilled watc1.,1rand gently dab oft the excesswater on to a cleanii-.

tissue;checkfor visible damageor contaminatio,nt-';of the glasselectrode(consulta memberof staffifthe glass is broken or dirty). Also checkthct the ';;'solutionwithinthe glassassemblyis coveringthe :",1

metalelectrode. ,i:'9H""'',,,

5, Calibrate the instrument: set the meter tomode,if appropriate, and then placethe alectrode

""

assernblyin a standard eolution of known FHi,,,,lusuallypH 7.00.This solution may be sr"rppliedse',,:ii:a liquid, or may be preparedby dissolvingameasuredamount of a calibration standard in ,l;1watericalibrationstandardsaro often providedin 'iitabletform, to'be, dissslvedin water to give a:'::l

of solution. Adiuet the ,Barticular volumscElibrationcontrol to give the correet readlng, ,'lRememberthat your cslibrstionstandardswill ';only give the specifiod pH at a particular '

temperature,ueuallyeither 20'C or 28-C, lf You',:*,are working at a different temperature,you mu$t ,;j

i

establish the actual pH of your cnlibration'from-.;i

standards, eithsr frorn tho supplier, or

literatureinformatisn.

, ..l

6. Removetho electrods assembly from thecalibrationsolution and rinre again with distlllsd rt;'

water: dab off the excesswater.Basicinstruments ' Ifurther calibrationstsps (single-point

hsve noealibratlon),while the mora reflnedpH meter*have',,.additionglcalibrationprocedurea" ,,'",,:

lf ycu are usinga baric inetrument,you shpuld+ireheckthat your apperatueis neeurflt ovsr ths' ':,


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appropriatepH range by meaauring the pH of

'

snCItherstandsrd whoee pH iE close to thatBxps0tedfor thetsst solution, lf thn etandsrddoeE.nntgivetheexpectedreading'thc ln$trumentin nntfunetioningcorrectly:esnnulta member of rtaff,

lf you are using an instrumentwith s slope'r,,eontrolfunction,this will allsw you to cCIrr ctfor

,any deviation in electrioel potontial from that .:

'

prsdictedby tho theoreticalrelationship{at *goC,bontinuail :.

'f'f


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changein pH of 1.00unit should result in achange in electricalpotential of 8g.16 mV) byperformlng I two-point calibration, Havingcalibratedthe instrument at pH 7.00,inrmersein asecondstandardat the same temperatureasthatof

thefirststandard,usuallybufferedto eitherpH 4.00or pH9,00,dependingupon the expectedpHof yoursample*,Adjuet the slope control until the exactvalue of the second standard is achieved

(Fig.24.A. A pH electrodeand meter calibratedusing the two-point method will give accuratereadings over the pH range from 3 to 11:laboratorypH electrodesare not accurateoutsidethis range, since the theoretical relationshipbetween electrical potentialandpH isvalid.

'r'.' , i::'

:,.':'. i .::

Fiq.24.2 TherelationshipbetweenefectricalporentialandpH.Ths solid line showethe responseof a calibratetlelectroclewhiletheotherplotsarefor instrumentsrequiringcalibration:t ha6 the cclrrectslope but incorrect isopotentinlpoint(calibration

controladjustmentis neecled);z has thB correcrisopotentinlpointbut inesrreet slope(slopecorrtroladjustmentisncoded).

7. Once the instrument ia callbrstod, mearura the pHof your eolution{e},makingsurethat the electrodeaesembly is rinsed thoroughly betweenmeasurements.You shouldbe particularlyawareof

thie requirement if your solutions contain organicbiologicalmaterial,e.g. soil, tissue fluids, proteingolutions,etc.,sincethese may adhere to the glaaselectrode ancl affect the calibration of yourinstrument.lf yourefectrodebecomescontaminatodduring use, check with a member of staff beforecleaning:avoid touchingthe surface of the glass

electrode with abrasive material. Allow sufficienttime for the pH readingto stabilisein each solutionbefore taking a measurement:for unbufferedsolutions, this rnaytakeseveral minutes, so do nottakeinaccuratepHreadingsdueto irnpatiencel

8, Aftar use, tha elestrode aeeembly muct not beallowed to dry out, Most pH electrodesshould bestored in a neutral solution of KCl, either bysuspendingtho assembly in o small beaker, or hyusing an electrodecap filled with the appropriatesolution(typically1.0moll 1KCIbufferod at pH7.0).However, many labssimplyusedistilledwster as 0storage solution, leadingto loss of ions from theintoriorof the electrode assembly.In practice,this


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meansthatpHelectrodesstored in distilledwaterwilltakefar longerto give a stablereadingthan thosestored in KCl.

9. $witch the meter to zaro (whereappropriatsl,butdo not turn off the power; pH meters give morestablereadingsif they are left on during normalworking hours.Problems {and solutions) includo: inaccurate

and/or unstable pH readingscaus6d hy cross-contamination (rinse electrode assembly withdistilled wator nnd blot dry between measure,ments);developmentof a proteinfilm on the eurfaceof the efectrode (soakin 1o/o w/v pepsinin 0,1moll-1HCIfor at leastan hour);depositionof organie or

inorganiccontaminantson the glass bulb (usean organic solvent, suchas aestone, or a solutionof 0.1 moll*1 disodium ethylenodiamine-tetraacatic

acid, respectively); drying out of the internal

referencesolutions(drain,flushsnd refill with fresh

solution,thenallowto equilibratein 0,1 moll"1 Helfor atleastan hour); cracksor chips to tho sudaca ofthoglassbulb(usea replacementslectrode).

Exercise:Briefly list (in pointforrn)theprocedurethatyouwouldfollow whenrecordingthepI{ valueof a solutionusingtheportablepH meterprovided.

(a) At the beginning of an enzymaticreactionbasedexperiment,wheremediumiscon$ideredas slightlyacidic.(b) At the end of theexperiment,wherephenotphthaleinindicatorhasturnedto apinkco

lor,


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Buffermlutloni

D{tnttlew One that reclrh aehangein H+ concentmtlon(pII) on acldlc or rlknli.

RatherthsneimplymeasuringthepHof a solution, youmay rr'idr tc CONTROLthepHinmatabolicexperiments medium solutionsor any other

orin agrountr forcellculture,biomolecules out the experiment.

through

Oneof the mosteffectivewaysto Eonffil pHiu to u$e a buffer solution* Inbriefthebuffsrsolutionis a mixture of a wcah ecld and its eonJugated will be

bru*. Added protonsneutraliredby the anionicbssewhile a reductionofprotons(ie.Additionofhydroxylions)will becounterbdansed ofasid.Thus, pairwill astas a

bydissoeiation theco4ir,rgate"buffer"topHchange,

resist due to the prscnceof eellular

Inlivingoystems,mostbiologicalsystems topHchanges

complexes cations,ssna,lroastflss substrate, andinhibitorofcertainentTrncs.

phorphatebuffersreadilyforminsoluble withdivalent whilephonphatoactivator

An ideal huffer far blatogleat purpaseswould pa sess the fellawingcfiaractenstics;

r impermeabtlltyta bHqlml mamfuianan;r

' ?:?H:::,",#i;Jx,,lti*'::[

rE:

lagicatprooasses,'

:;,

#ilff!:#;ii, ::

r:,:::,::'

,',:,?:ffi mpoattio


19/31

:i'l*i:#':ifi*e co n o r

saltconoontration;. Ilmited pH changa ln rasponsatotamperatura,

tts4S67g

pH:rig,.i,,"

UsefulpH rangesof somecommonlyusedbuffers


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tomixstoeksolutionsofaaidisandbasie

TnbleI For traditional buffers,it iscustomarysomponents togivetlrerequired

in the conectproportion pH,

Volumeof Vofumoof

stock(al stock(b)RequiradpH NarHFOI NaHaP r(at?5'Cl (mll (mll

6,0 0.2 {g.g

4.2 9,3 40,76.4 13.3 9fi.78,6 18.8 31.26,8 21.5 25.67.0 30.6 19,67,2 30.0 14.07,4 40,5 9.57.6 43.6 6.57.8 45.8 4,2

8.0 47,4 2,6Romember:Buffersolutionswill only workeffectively if theyhavesufficientbuffering capacityto rcsist the pH expectedduring the courue of the oxperiment. Weakbufferswill notbe able to buffergrourthmediaof dense suspensions

of c llsfor moffe thana few minutes,

Finally: when preparinga buffer solution using tabulated information,always confirm thepH with apH meterbeforeusage.

lo


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3, Botlc Skilh lnrtrlicruseopy(eallbrotion,mGetuncncnts,

ond AAlcrcmctry

drawirgr qndphotegrqphy)

Neverassumethatpreviouspersontou$eyourmiuoseopehasleft it set up conectly.Apartfromdiffereneesin user's eyes,the miuoscope needstobeproperlyset up for eachlens combinationused.

Bastepaftsofa ilfleroscope &, Objecttve lens,

.Diagramof the Olympue binosular

*+

microrcspemPctetCX41"

rThe lanrpin the base of tho 6tand(1)supplies

light; its brightnees ie controlledby an on-otfswitch and voltage control (2). Never usemaxirnumvoltogeor the life of the bulb will bereduced* a settingtwo-thirdato thrae-quarter$of maximum should be adequatefor mostspecimens.A fiald-irisdiaphragm may be fittedcloso to the lamp to control the area ofillumination(3).

rThe condensercontrol focuses light from thecondenserlens system(4) on to the specimenand projectsthe specimen's image on to the

front lens of the objective. Correctly used, itsnsuresoptimalresolution.

rThe eondenser-iris diaphragm(5)controls theamount of light entering and leaving thecondeneer;its aperture can be adjusted usingthecondenser-irisdiaphragm lever (6).Use thisto reduceglareand enhance image contrast bycuttingdownthe amount of stray llght reachingtho objectivelens.

r

Thespecimen(normallymounted on a slide)isfitted to a mechanicalstage or slide holder

(7) using a spring mechanism.Two controlsallowyou to movs the slide in x and y planes.Vernierscales(seep. 134) on the slide holdercan be used to returnto the same placeon aslide. The fine and coarse focus controls(8)adjustthe height of the stag relative to thelens systems.Take care when adjusting the


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focuacontrolsto avoid hitting the lenseswiththe stsge or slide.rThe objective lEne (9) supplies the initialnragnified image; it is the most importantcomponent of any microscope because itsqualitiesdetermina resolution,depth of fieldand optical aberrations.Theobjectivolensesareattachedto a revolvingnosepiece(10).Takscarenot to jam the longerlenseson the stageorslide as you rotatethe nosepiece. You should

magndcnlhn

feel a distinct click as each lens is moved intoposition.The magnification of each objectiveiswrittenon its side; a normalcomplementwouldbe x4, x10, x40andx100(oilimmersion).

rTheeyepiece lens (11)is usedto furthermagnifythe image from the objectiveand to put it in aform and position suitable for viewing. lts

magnificationis writtenon the holder (normallyfrncfEc0F

ruba ln90l

x10).By twisting the holder for one or both ofthe eyepiece lensesyou canadjust their relativeheights to take account of optical differences

haErir6rdy

betweenyour eyes. The interpupillarydistanceliC. Objecrive len$ paramelers. Most

scale {12) and adjustment knob allow lensosarairrscribcd

to$howrhgdersilslabelteJcompensationto be made for differenceein the above.Th numoricalaFertur is a rnoasure of

lhc light-grtnsfing

powerof the lens.

distance between users' pupils.

ft


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Cafibrattngan EyePteeeMteranutu&AddtngLlaearScaleto Drewlngs

* otor

li' Uhe {"$e I

Adting linear scalerto drawings '..

Themagnificationof a light-microscope

imageiscalculatedhymultiplyingthe objectivemagnificationby theeyepiecemagnification.However,themagnificationof theimagEbearsnocertainrelutionto themagniftcationofanydrawingof theimage-youmayequallywellchooseto drawthesameimagel0mm or l0cm long.For thisreason.rl isessenrialtaadda scnletoatl yourdiagrams,You eanprovideeitherabargivingtheestimatedsizeofanobjectof interc$t,or a barof defincdlength{e,9.100Fm).

Thesimplcstmethodof estimatinglineardimensionsisto comparethesizeof theimageto thediameterof thefield of view.You canmakearoughEstimateof thefielddiameterbyfocusingonthemillimetrescaleofa transparentruler using the lowestpower objective.Estimatethediameterof thisfielddirectly,thenusetheinformationto work out the

r,?.S) Calibrating micrometerfielddiameters example,if thefieldatan

aneyepieee atthehigherpower$lra retet,ForAlignthe two scalesand readtha

{graticule). overallmagnificationof x40 is4mm, at an overallmagnilicationof x 100

ofstagamiorometBrnumbsr divisione = mm(1600pm).

91?11 it will--ue:+o1to0x 4mm 1.6

a particular number of eyepiece-

ln colour) for ;__.--.. r^^ r-.-:_^,,c _ -.^_:^^-_j^--_rr^_ /..-_+:_..r-\

Createraccuracycancancan DeoDlalneo lI ananan eyeplesemlcromstGt (grallculel

(-treater ACcUfACy beobtainedif eyepiecemicrometer(graticule)

mi*omotermi*omotermi*omoter divi*ions

divi*ionsdivi*ions {ghown{ghown{ghown ininin bl;'.i;'l;'J*blackl. In'l*this cgss26,gecatedivisionaof o.oirr pis used.This carriesa finescaleand hts insidean eyepiecelens.Thel*,2,ri9rhmlareoqulvatontto 100eyepieceleyepiecemicrometeris calibratedusinga stagemicrometer,basicallyadivisiona'8o aach eyopiecedivisionI slidewithafinescaleonit,Figure{A),lhowshowtocalibrateancyepiece


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* 0'00265 2'86pm'This0'286+100 mm*

-':^:I micrometer,----^.-.^ alongwithaworledeximpte.oneeyouhavecalibratidyour

typicslvalue of-' x400

fora totalmognlflcation^-*-J|

eyepieeemicrometerfor Eachobjectivelensused,yCIucanxseit toqe.e*trg

(e,9.(6,9.xq0objectlvo ayepiaca).

x40oblectlvoandandandxxx101010syepiaca).objects:in theexampleshownin Fig.'(A]9. rcalereadingisrnultiplied

inmicrometles. thewidth

so,if youmeasured

llclngan eyaplengratlcule-ahaosa by2.65pmtogivethcvaluemicrometer to

in, Tvipie\a-uns'carieiBonitni-i ofahumanhairat34eyepiece units,thenthiswillbeequalyoaratrongarayeandchtick 34x 2.65-=90.1 approachisto puta scalebarsn a

tha{you gm.An alternativehavemadetha corraetadjustmgnqsto diagram,e.g.a 100pm scalebar wouldbe equivalentto the lengthof

'

the eyepiecalenseeag detalledon u

alrnost3geyepiecemicrometerdivisions.

Nof*si\A/aA/l"'{'*fvorun f' r'q , A , 'A**+;{l Irtr{ {*ofra!*g:-T?psc*'{s -"*tln',f *f gi*tr''{l**l **uo**.fu..u" (3*p u*r'{ (Scodr } rn.',r:i*'vt14h 4qr;.t+r-{ t* fl! ' {J| *: r"?.

r7


25/31

CellPinsr+Jqlfln{ Tiss,uFDiaqrans{,Mans)

Gl KEy POTNT You maynat feal confldantabout being aAfeffiIt eloducl quallty arfianrkin praetfcals,aspeelaltywficn .ffi

-

tlma allawcdla tlmttard, Howaver,tha raqulrementsef eiotefficaldrawlngarenataadamandlngf,$fou mightthinkend#ffi*ktll* requlrad aanbe laarnad,By feltawlng theguldatlnaeaffitachnlque*sxplainedhalow, rnosf sfudpnfr shouldba abla,ffirproducagooddiagrams, ,it

ffi

--H

Themaintypesof figurerI

Celt diagrnms (4 )In a cell diagram, your aim is to show acsur&tely the details of thd

individualcells in a tissue" You wnuld normally^draw

a cell diagram from,.

a *pecimen viewed by light microscopyat e magnificationo? ?00x oi'tnsre,Your dingram shouldbe detailecl, butneednot eomprise morstha.[:ja few cells, especi*lly if they are all similar(Fig"" (a)),you may bd_i

ssked to drawonlyone complete cell,plusparrnf any neighberuring

cells;j

so that the interrelationshipbetweencells can be seen. Uselabels to note..any structuresthat are stained,and what this.nrenns in tsrms of cell,l.ehemistry.For cellswith thin walls,jou mnyneedto exaggeratethewall '

thicknenrto show it on your diagram.If this is necessaryo

add aniexplnnatorynoteto ysurdiagram(seeF'ig,

,!l)t.

Tissue diagrams or msps ( lr ) * Lu )

Thepurpoieof a tissuediug.u* is to showher*the tissues areplacedina section through a wholeorganismor one of its organ$(compareFigs ' (b)and' (c)).Youshouldnot inuludc any cellular detail unlessspecificallyinstructed,and shadingor hatchin[ should be kept to aminirnum,The mnin difficulty you will eneounteris deciding where todrnw the boundnryline hetween tissues:cell differentiationis rarelyeliscrete, of bothtissues,

soeellsat the boundarymay show eharacteristics


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A ccrtain amCIuttt of trnckgrnundknowledgeand interpretive skill isrequiredfor this.

ll{orphologicnl dicgt'cttnffand hodl' plansLdh {*:ln a morphologicaldiagram,the objective is to provide a lifelikerepresentaiioninOieatingthe main surface features(Fig.{.'i. For abodyplan,youraimis to show the relationshipsbetween organ$

segrRents'or oth r body parts,often following a dissection (Fig.f, "r.In both cases'shadingshouldbe avoided,unlessit servesto highlight a particularfeature.You should fully labelbothtypesofdiagramandaddnoteswhere

relevant.The main problemyou arelikely to encounter is keepingthedifferentpartsin proportionto each other* thiscan be solvedhy usingconstructionlinesand frames (seebelow).

,Wnen:drd*lng iati :aiagrams'tiesua

msp$ and bodYPlans,it mlght halPto

beir in mind the followingdefinitiona:

Galh *,',the 'AriftOtng

blocks'of life.

Alwaycaepsratedfrom thelr environ

ment and othsreollehYa mambrene *

a cell watl.Tvpiealdimensione:'10to

100pm.Example*r, call;

laafmetrophyllkidneVnephroneslhYssstcell.Tlrru*l'-eollantloneaf oellehavlng

.ind

aimllarilructure frlnctl'on,Fromone cell thlsk to msny cell laYers.

Examplen;pallaademetoPhYllin afaaf;rsnal'cortex;lnlthakidneY'-

organr struetureE conrloting ofaeveral tiaeues workinga3 a grouptopedorma apeciflc function.Examplae:leaf;,kidney. .:.

t4


27/31

moryhohglcg'l dhgirm -alnea ffeaasaneaof thta typaof dnwlng la to ehawonly tha lmportant featuna, lt la vnrthmakinge llst af the ftemato be lneludad,er te hlghllghtthaea when mentlend lnyour Braxieataehadula,Eon't furgathotrevsr,to add anough anatemleal

datalt,a,g, af a bodV plan,, ta ptaeeeutllna&afureefn the approprlatte

eontexf,Alwayeadd a gr"eleandafbeflrrata&lltng, Netethat lf pu urfrh b chour ftna datat[,conaiderdalng fhh en'a separ fa hfghepecaladiagnm,

frl (cl

avtiale upper a.viddrmi*

\\

-\\

p*llsedaffiosapltyll(2 layrra)

II-

,T,T6wqy

.*-modopPtyll

phloarh Xffi\

lawr*#afl&rmla

:ffiffiaw.,x

awms #ub-gtamawlbaundad by *tr &tdl6apeir of quad callo

Try,nswt.rcxedon af a dtea6idan brt

wetefabaaryneymcaophyllccllTg

Fig' Celland tissuediagramecomparedwith a photomicrograph.(E)CElldiagramshowingrepreseRtstive

*pongy mesophyllcell* from an electron mieroacope image of a leaf eeetion;tb) aphatorni*ographef a transveree eestiqnthrough a leaf {light


28/31

microscopyl"

Oifferentecalesare used, but in beth easesthay ara indieated uy a ber {seep, 2Sg}.Nlntehow the eell"wEll etructurs i$clsrifiedin {a} by slightly exaggeratingits thicknees.In a light microscopesectiontessdetail of organelleswoutd be visihle,

{c}Tissuediagramof port of a T$ leafsection.Notethst this diagramis unclutteredby detail and tha inclividufitticsus$ ars notshaded* the $caleis indieat&dby a magnification factorrelevantto the actualsireof the diagram {r*ctueedhere}(sasB"l5g).(4)rlbntshIrifo$lhl|c

ospodlrpffiufT llaf

T

l,*

l*

I

co$ifu$*l{efnet nrnr*rqfirf

(d).Frrphotosicet

diasfonrof a French

f,t prtaseotuEvulsarisL, Nots t;e

:.:in_.:Tdllp,

lnefof ohadlngcomparedwith Fig.1g.4.

j:n:^*L,g-)-::lfrtc or a borJy

pron.rhh ia a dia rsftor

gsn rnrdissooronof oneanhworrn,tu*rbricusfalrJitristdortrtview.Nst rh! tack ofchadingilil;

ril';fr;

and{nnotationgtaidsntifyefoorly

wtritt a*nuurlOr"i*n"

ttf


29/31

LaborrtoryExcrcire:Callbrrtlonoftheeyepiecegratlcule

(i) Outliningtheprocedurethatyoufollowed,calculatethe value foreach eyc piecedivioionat X 100magnifieation.(ii) Calculatetheaverageheigbt of thepalisadelayerof cells in the leaf cmss sectionprovidedtoyou.(iii) Drau'a lined diagram(map)of the TS leafreetionprcvided.(iv) Drnw a cell diagramONLY for a few mesophyllcellsin ttre cross seetionoftheleafgiventoyou,

acoll?

n o

toomuchunnemrury drtrll

?

\\F

hengingllnrr

ttpEnetratinglins

poor,llntchy

llnequalltyalo

t//

cmurlly

il0

dra$ndfirll

$ll

t// g

Fig, i Exampleaof ';commonErrors inbiologicaldrawings.Moctofthffe mistakasaredueto lack of car0 or etteffiion to d tsil * eaeilysolvedl l

t5


30/31


31/31

lab skills full lecture scanned

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