biology practical skills booklet

7/24/2019 Biology Practical Skills Booklet

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Biological Sciences Important Practical Skills

1. Carrying out basic laboratory techniques and understandingthe principles that underlie them

2. Working saely! responsibly and legally in the laboratory! "ithdue attention to ethical aspects

#. $esigning! planning and conducting biological in%estigations&. 'btaining! recording! collating and analysing biological data(. )sing data in se%eral orms e.g. numerical! te*tual! %erbal and

graphical+. ,%aluating your e*perimental technique

-eneral pproach to Practical Work

/ead handouts in ad%ance! "here possible! so you understand"hy you are doing a particular practical and the principlesbehind it

Be a"are o the time in "hich you ha%e to "ork Consider saety ha0ards beore you begin 'rganise your "orking bench space Write up "ork as soon as possible ater the practical

ccuracy and Precision in techniques

ccuracy the closeness o a measured data %alue to its true %alue

Precision the closeness o repeated measurements to each other

So a balance "ith a ault in it could gi%e precise 3i.e. %eryrepeatable4 results but inaccurate 3untrue4 results. )nless thereis a bias 3ault4 in the measuring system! precision "ill lead toaccuracy.

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Bias can be due to56 Incorrectly calibrated

instruments e.g. aulty "ater

bath ,*perimental manipulations

e.g. using a thermometer tomeasure temperature canitsel decrease thetemperature

Sub7ecti%e ideas by thee*perimenter e.g. 7udging

"hen an end6point is reached!or i*ing results to it thosee*pected

8inimising ,rrorsWhen designing an e*periment56

,nsure that the independent%ariable is the only ma7or

actor that changes Incorporate a control

e*periment to sho" it is onlythe independent %ariable"hich causes the measured

eect Where appropriate! select e*perimental sub7ects randomly to

cancel out %ariation arising rom biased selection 3this isimportant in ecological in%estigations4

9eep the number o replicates as high as possible ,nsure the same number o replicates is done or each %alue

o the independent %ariable

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Identiy other actors "hich could aect the dependent%ariable and keep them constant 3control %ariables4 e.g.temperature! %olumes o solutions! light intensity! time orreaction

8inimising 8easurement ,rror Common source is carelessness e.g. reading a scale in the

"rong direction: reduce this by more careul recording 3thinkho" you can do this4 and by repeating the measurement

ccuracy depends on using suitable equipment"ith care.

ccurate measurement o liquids ;igh %iscosity liquids are diicult to transer: allo" time or

all the liquid to transer 'rganic sol%ents or hot liquids may e%aporate quickly! making

measurements inaccurate: transer these liquids quickly andco%er containers


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)sing balances >e%er "eigh anything directly onto a balance?s pan: this "ill

contaminate it or other users. )se a "eighing boat or slip oaluminium oil! or paper

>ote reading to 2 decimal places. >.B. When calculating amean a%erage o readings! the a%erage should also be to 2decimal places

Potential errors5 samples spilt onto the pan "ill be measuredbut not used: as "ill samples let i the "eighing boat is notscraped clean. ;o" "ould the results be aected=

8easuring and Controlling @emperature

;eating samples5o Wear saety glasseso )se a thermostatically6controlled"ater bath i possible

and suitable: check the temperature using athermometer: do not rely on the temperature sho"n onthe dial

o >ote that i you are not using a thermostatically

controlled "ater bath! this "ould be a good

impro%ement you could mention in an e%aluationo State the temperature used and the time or "hich

heating is carried out e.g. Benedict?s test heat or 5-10 minutesin a water bath at 80oC

o )se insulation i necessary or possible Potential errors5 has the temperature %aried rom the stated

%alue= ;o" "ould this aect the results=

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8easuring @ime )se a stop "atch rather than a clock 8ake sure you kno" "hich buttons to press beorethe

e*periment startsA

>ote time readings to a suitable number o decimal places.>.B. When calculating a mean a%erage o readings! thea%erage should also be to the same number o decimal places.Could you actually measure the time this accurately=

Potential errors5 ho" easy is it to kno" "hen to start or stopthe timer= What dierence "ould this make to the results=

Preparing $ilutions

Solutions are usually prepared "ith respect to their molar concentrations 3moll or moldm#4 3a mole is a gi%en

number o molecules o a compound: 1 mole has a mass ingrams equal to the relati%e molecular mass o thatcompound4 or

mass concentrations 3gl or gdm#4

Both these are the amount o a substance per unit %olume o

solution. i.e.

Concentration mount olume

Concentration must al"ays be gi%en units.

In practicals! you "ill oten be gi%en stock solutions to use. @heseare solutions o kno"n concentration and are %aluable "hen makingup a range o solutions o diering concentrations. @hey also sa%e"ork i the same solution is used o%er a long time e.g. a nutrientsolution. Stock solutions are more concentrated than the inalrequirement and are diluted as appropriate.

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Preparing a dilution series series o dilutions is %ery useul or a "ide range o procedurese.g.

to in%estigate changing the concentration o substrate in an

en0yme reaction to prepare calibration cur%es or colorimetry

How to make a dilution series: Always start with the most concentrated solution

1. $ecimal dilutions

,ach concentration is one tenth that o thepre%ious one 3log1Ddilution series4

8easure out the most concentrated solution "itha 1DE e*cess

8easure one6tenth o the %olume required into a

%essel containing nine times as much diluting liquid

6

8ost

concentratedsolution 3ine*cess4

1 ml 1 ml 1 ml 1 ml 1 ml

)ndiluted31D4 11D31D614

11DD31D624

11 DDD31D6#4

11D DDD31D6&4

11DD DDD31D6(4

F mldiluent


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8i* thoroughly

/epeat to obtain concentrations 11D! 11DD!

11DDD! etc times the original @o calculate the actual concentration o solute

multiply by the appropriate dilution actor

2. $oubling dilutions56 ,ach concentration is hal that o the pre%ious

one )se t"ice the %olume required o the irst! most

concentrated solution 8easure out hal o this %olume into a %essel

containing the same %olume o diluting liquid 3e.g.distilled "ater4

8i* thoroughly

/epeat or as many doubling dilutions as arerequired

@he concentrations obtained "ill be G! H! ! etctimes the original

Potential ,rrors 3/emember thinking about these can help youe%aluate your procedure4

Contamination rom syringes: rinse bet"een use or use ne"syringes or each solution to a%oid carry6o%er o solutions othe "rong concentration

3>ote that "hen transerring a range o prepared dilutions

rom sample pots into test tubes! you should start "ith thelo"est concentration and! i you rinse the syringe in the ne*t

concentration beore dispensing it! you can use the same

syringe or pipette4

Inaccuracy in measuring %olumes: any slight inaccuracy "illlead to compounded inaccuracies! so the most dilute solutions

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ha%e huge errors in concentration 3see precautions inmeasuring liquids abo%e4

I@S I> @;,

B'$ 'J @;, @BB take care iusing the computer as it sometimes automatically changesthese4

'bser%ations o results 3not your interpretation o themA4Write these in as much detail as possible

/epeated readings: use a separate column or each @hink about any additional columns you may need! and dra"

them in at the start. dditional columns may be used to sho"step by step calculations e.g. %olume 3cm#4! time 3s4! 1time31s4! rate 3cm#s4

Calculated mean a%erages 3$o not use a greater number odecimal places than you ha%e in the ra" data4

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$esign your table to make the recording o data asstraightor"ard as possible! to minimise the possibility omistakes

,*plain any unusual data %alues in a ootnote: don?t rely on

memoryA ,.g. orgot to start stop"atch

Colorimetry

@he spectrophotometermeasures the absorptiono radiation in the

%isible and u% regions othe electromagneticspectrum. @hespectrophotometerallo"s precisemeasurement at a particular "a%elength. colorimeter is simpler!using ilters to measure broader "a%elength bands 3e.g. green! redor blue light4.

Principles o light absorption

@he absorption o light is e*ponentially related to thenumber o molecules o the absorbing solute in the solutioni.e. KCL! solute concentration

bsorbance at a particular "a%elength is oten sho"n as asubscript e.g. ((D absorbance at ((D nm

@he proportion o light passing throughthe solution is kno"nas transmittance 3@4 and is calculated as the ratio o the

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emergent and incident light intensities. It is usuallye*pressed as a percentage

@he colorimeter has 2 scales56o n e*ponential scale rom 0ero to ininity! measuring

absorbanceo linear scale rom D 1DD! measuring 3per cent4

transmittance Jor most practical purposes you should use absorbance! "hich

is linearly related to the solute concentration KCL

,*tension inormation

Absorbance (A) is i!en by:-

log1D3IoBI 4

)sually sho"n as *! "here * the "a%elength in nanometreslso56

l[C]Where5 = a constant or the absorbing substance 3absorption coeicient4

l the length o the light path through the absorbing solutionC concentration in moll or gl

Calibration Cur%esBy preparing a set ostandard solutions!each containing akno"n amount orconcentration o asubstance! and thenmeasuring theabsorbance o eachsolution! a Mcalibration cur%eN or Mstandard cur%eN can be produced.

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3>ote that at high concentrations! the relationships abo%e do nothold true! and the straight line relationship sho"n may becomecur%ed.4 @he line can be used to estimate concentrations o solutein a test or unkno"n sample.

;o" to use the colorimeter1. S"itch on2. llo" 1( minutes or the lamp to "arm up and the instrument

to stabilise#. Select the correct coloured ilter 3It is best to use the

ilter "hich selects the range o "a%elengths most stronglyabsorbed by the sample because this "ill gi%e the ma*imum

reading4. @he most suitable ilter colour is usually thecomplementary colour to the solution being tested56

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. Check the reproducibility o the instrument: measure theabsorbance o a single solution se%eral times during analysis.It should gi%e the same %alue

Inaccuracies may be due to56 Incorrect use o cu%ettes

i. $irtii. Jingerprintsiii. @est solution on the

outside o cu%ettes Condensation on cold samples

3allo" cold samples to equilibrate

to room temperature4 ir bubbles in samples 3tap

gently to remo%e4 Insuicient solution 3reraction

o light at meniscus4 Cloudiness o sample 3decant o

supernatant to test! aterallo"ing precipitate to settle4

8icroscopy

Problems in light microscopy and possiblesolutions56

"o imae# !ery dark imae microscope not s"itched on ob7ecti%e nosepiece not clicked into

place o%er a lens lamp ailure

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$mae blurred and cannot be %ocused dirty ob7ecti%e dirty slide slide upside do"n

slide not completely lat on stage ine ocus at end o tra%el

&ust and dirt in %ield o% !iew eyepiece lens dirty ob7ecti%e lens dirty slide dirty dirty on lamp glass or upper condenser lens

Setting up and using the light microscope1. Select lo" po"er lens. 8ake sure the lens clicks into

position.2. ,*amine prepared slide "ithout the microscope and note

position! colour and rough si0e o specimen.#. Place slide on stage! co%erslip uppermost! %ie"ing it

rom the side. Position it "ith stage ad7ustmentcontrols so that the specimen is lit up.

&. Jocus using irst the course and then the ine ocusingcontrols. )se both hands to alter the ocusing controls:this helps keep the controls "orking properly and notgoing out o alignment.>ote5 @he image "ill be re%ersed and upside do"n "henseen by %ie"ing the slide directly.

(. Jor higher magniications! s"ing in the rele%antob7ecti%e lens careully checking there is space or it.d7ust the ocus using the ine control only. I theob7ect is in the centre o the ield o %ie" "ith *1Dob7ecti%e! it should remain in %ie" "ith the *&Dob7ecti%e.

+. When you ha%e inished using the microscope!

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@urn the ob7ecti%e lens back to *1D


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8ethods o estimating si0e

1. Compare the si0e o the image to the diameter o the ield

o %ie"56a. Jocus on the millimetre scale o a transparent ruler!

using the lo"est po"er ob7ecti%eb. ,stimate the diameter o this ield directlyc. )se the inormation to "ork out the ield diameters o

higher po"ers e.g. i the ield at *&D is & mm! at amagniication o *1DD "ill be &D1DD * & mm 1.+ mm

2. -reater accuracy can be obtained i an eyepiece graticule

3micrometer4 is used56a. -raticule carries a ine scale and its inside an eyepiece

lensb. -raticule is calibrated using a stage micrometer i.e. a

slide "ith a ine scale in it 3ou "ill do this yoursel4.c. 'nce you calibrate your eyepiece graticule or each

ob7ecti%e lens used! you can use it to measure ob7ectsd. In the e*ample belo"! the scale reading is multiplied by

2.+( Qm to gi%e the %alue in micrometres.e. %oid putting too many signiicant igures in any

estimates o dimensions: there may be quite largeerrors in%ol%ed

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'ecordin by bioloical drawins

@he making o accurate dra"ings is an essential skill or d%ancedle%el biology students. Students are required to make dra"ings o

e*ternal eatures o "hole specimens! parts o specimens!dissections and prepared slides.

$ra"ings must be done on white aer usin shar encil.Students should note the ollo"ing points5

1. dra"ing should be genuine and accurate record o "hat hasbeen seen. $o not copy diagrams rom books "ith littleresemblance to the specimen! dissection or prepared slide

under in%estigation.2. Jor lo" po"er dra"ings! only the outline o the structures or

tissues needs to be dra"n. Indi%idual cells are required onlyor high po"er dra"ings.

#. $ra" "ith bold! dark! smooth lines. Shading and the use ocrayons are not encouraged.

&. %oid making dra"ings on both sides o a paper.(. In making a dra"ing! irst decide "hat you "ant to sho".

@hen plan your dra"ing to it on the page. It is importantthat the %arious parts o the structures are dra"n inproportion.

+. $ra"ings should be large! neat and tidy.O. ll rele%ant structures should be ully and clearly labelled

usin encil ,ach label should be connected to theappropriate part o the dra"ing by a clear labelling line. @he

labelling lines should run as hori0ontal as possible and shouldnot intersect "ith one another. $o not label too close to thedra"ings! and do not "rite on the dra"ing itsel. $istributethe labels "ell around the dra"ing so that the labelling linescan be kept reasonably short.

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. ,%ery dra"ing should include a title! the magniication po"er!and the plan o %ie" o the specimen 3such as trans%ersesection! longitudinal section4.

F. 8ake additional dra"ings i important details are too small to

be sho"n in a lo" po"er dra"ing. @his can be done by makinga simple dra"ing o the main eatures! and other dra"ings ondetails o small parts only. Jor e*ample! the recording o atrans%erse section o a stem should include a lo" po"er plano the section and high po"er dra"ings o dierent cellstypes as required sho"ing the detail o the cells.

1D. I suices to sho" the internal structural details in asmall representati%e sector o the specimen "hich sho"s

repetiti%e eatures.11.It is sometimes appropriate to make short succinct notes

close to the labels. Such annotated dra"ings are particularly%aluable as they combine a record o structures "ith relatedunctions andor biological interests. ou may "ant to labelan organelle to describe staining e.g. nucleus 3darkly stained4:starch grain 3blue6black4

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&rawin *rahs

-raphs gi%e a %isual impression o the content and meaning oyour results. @ables pro%ide an accurate numerical record o

data %alues. -raphs should56

o include all material necessary to con%ey the appropriatemessage "ithout reerence to the te*t

When dra"ing graphs56o Collect all data %alues to be plottedo Consider "hether a graph is the best "ay to present

the data

o Choose a concise e*planatory title to establish thecontent

o Consider the layout and scale o the a*es careullyo )se the * a*is or the independent %ariableo )se the y a*is or the dependent %ariableo When neither %ariable is determined by the other! or

"here %ariables are interdependent! the a*es may beplotted either "ay round

o ,ach a*is should ha%e a descripti%e label sho"ing "hatis represented and including units o measurement!separated by or "ritten in brackets.

o ,ach a*is must ha%e an appropriate marked scale!sho"ing the location o all numbers used. Jill thea%ailable space on the paper

o I scale breaks are used! sho" them clearlyo Choose the symbols or each set o data points! i

plotting more than one set o datao Include a key to symbols o dierent data sets i

necessary

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o @o plot %ery large or small numbers! the plotted %aluesmay be measured numbers multiplied by a po"er o tene.g. 1D6#* population o bacteria mm#

o $ra" a trend line or each set o points5

Cur%e= Straight line=

Points 7oined "ith a ruler= 3@his is really only %alidi there is 0ero error. i.e. all the plotted points areprecise and accurate4

o l"ays dra" the simplest line that its the datareasonably "ell and is biologically reasonable.

o @ry to a%oid the need to e*trapolate plotted cur%es bybetter e*perimental design. 3@his could be a point ore%aluation o an e*periment4.

o >e%er allo" a computer programme to dictate "hat agraph looks like: make sure you can alter scales! labels!

a*es etc and make appropriate selections. $ra" cur%esreehand i necessary.

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$nterretin *rahs + Analysis and ,!aluation

1. $escribe the relationship bet"een the %ariables! quotingdata.

2. ,*plain "hat the relationship means "ith reerence to thebiological principles in%ol%ed.#. Consider the content5

a. What "as the aim hypothesis o the in%estigation=b. Why "ere the obser%ations made=

&. Consider "hat kind o graph is presented: is it an appropriatechoice or the data=

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a.


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%alidity o the line. straight line implies one actor%aries in direct relationship to another: the truesituation may be more comple* e.g. an e*ponentialrelationship could be more correct.

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biology practical skills booklet

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