Essential A level Biology Practical Skills
What you need to be able to do:
1. Carrying Carrying out basic basic laborato laboratory ry techniques techniques and and understand understanding ing the principle principless that underlie them 2. Working safely, responsibly responsibly and legally in the laboratory, laboratory, with due attention attention to ethical aspects 3. Designing Designing,, planning planning and conducti conducting ng biological biological inestigat inestigations ions !. "btainin "btaining, g, recording, recording, collatin collatingg and analysing analysing biologica biologicall data #. $sing data data in seeral forms forms e.g. numerical, te%tual, erbal and and graphical graphical &. 'aluatin 'aluatingg your your e%peri e%periment mental al techn technique ique (eneral )pproach to *ractical Work •
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+ead handouts in adance, where possible, so you understand why you are doing a particular practical and the principles behind it e aware of the time in which you hae to work Consider safety ha-ards before you begin "rganise your working bench space Write up work as soon as possible after the practical )ccuracy and *recisio *recisionn in techniques
)ccuracy the closeness of a measured data alue to its true alue *recision the closeness of repeated measurements to each other
/o0 a balance with a fault in it could gie precise i.e. ery repeatable results but inaccurate untrue untrue results. $nless there is a bias bias fault in the measuring measuring system, precision will lead to accuracy. ias can be due to4 • 5ncorrectly calibrated calibrated instruments e.g. faulty water bath • '%perimental '%perimental manipulations e.g. using a thermometer to measure temperature can itself decrease the temperature • /ub6ectie ideas by the e%perimenter e%perimenter e.g. 6udging when an end4point is reached, or fi%ing results to fit those e%pected 7inimisi 7ini mising ng 'rror 'rrorss When designing an e%periment4 'nsure that the independent ariable is the only ma6or factor that changes •
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Essential A level Biology Practical Skills
5ncorporate a control e%periment to show it is only the independent ariable which causes the measured effect Where appropriate, select e%perimental sub6ects randomly to cancel out ariation arising from biased selection this is important in ecological inestigations 8eep the number of replicates as high as possible 'nsure the same number of replicates is done for each alue of the independent ariable 5dentify other factors which could affect the dependent ariable and keep them constant control ariables e.g. temperature, olumes of solutions, light intensity, time for reaction 7inimising 7easurement 'rror Common source is carelessness e.g. reading a scale in the wrong direction9 reduce this by more careful recording think how you can do this and by repeating the measurement )ccuracy depends on using suitable equipment with care . •
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)ccurate measurement of liquids •
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:igh iscosity liquids are difficult to transfer9 allow time for all the liquid to transfer "rganic solents or hot liquids may eaporate quickly, making measurements inaccurate9 transfer these liquids quickly and coer containers ;iquids likely to froth e.g. yeast or protein solutions are difficult to measure9 transfer slowly /uspensions e.g. yeast or cell cultures may sediment9 mi% well before transferring $se measuring cylinders on a leel surface so the scale is hori-ontal9 fill to below the desired mark, then add liquid slowly e.g. by pipette to reach desired leel 7ake sure there are no air bubbles in syringes when measuring olumes. '%pel liquids slowly and touch the end of the syringe on the essel to remoe any liquid stuck to the end *otential errors inaccurate measurements for reasons gien aboe. What effects would this hae on the results< $sing balances
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=eer weigh anything directly onto a balance>s pan9 this will contaminate it for other users. $se a weighing boat or slip of aluminium foil, or paper =ote reading to 2 decimal places. =.. When calculating a mean aerage of Page 2 of 16
Essential A level Biology Practical Skills
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readings, the aerage should also be to 2 decimal places *otential errors samples spilt onto the pan will be measured but not used9 as will samples left if the weighing boat is not scraped clean. :ow would the results be affected< 7easuring and Controlling ?emperature
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:eating samples o Wear safety glasses o $se a thermostatically4controlled water bath if possible and suitable9 check the temperature using a thermometer9 do not rely on the temperature shown on the dial o =ote that if you are not using a thermostatically controlled water bath, this would be a good improement you could mention in an ealuation o /tate the temperature used and the time for which heating is carried out e.g. enedict>s test @ heat for 5-10 minutes in a water bath at 80oC o $se insulation if necessary or possible *otential errors has the temperature aried from the stated alue< :ow would this affect the results< 7easuring ?ime
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$se a stop watch rather than a clock 7ake sure you know which buttons to press before the e%periment startsA =ote time readings to a suitable number of decimal places. =.. When calculating a mean aerage of readings, the aerage should also be to the same number of decimal places. Could you actually measure the time this accurately< *otential errors how easy is it to know when to start or stop the timer< What difference would this make to the results< *reparing Dilutions
/olutions are usually prepared with respect to their molar concentrations molBl or molBdm 3 a mole is a gien number of molecules of a compound9 1 mole has a mass in grams equal to the relatie molecular mass of that compound or mass concentrations gBl or gBdm 3 •
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Essential A level Biology Practical Skills
oth these are the amount of a substance per unit olume of solution. i.e.
Concentration
)mount olume
Concentration must always be gien units.
5n practicals, you will often be gien stock solutions to use. ?hese are solutions of known concentration and are aluable when making up a range of solutions of differing concentrations. ?hey also sae work if the same solution is used oer a long time e.g. a nutrient solution. /tock solutions are more concentrated than the final requirement and are diluted as appropriate. *reparing a dilution series ) series of dilutions is ery useful for a wide range of procedures e.g. to inestigate changing the concentration of substrate in an en-yme reaction to prepare calibration cures for colorimetry • •
How to make a dilution series: •
Always start with the most concentrated solution
7ost concentrated solution in e%cess
1 ml
1 ml
1 ml
1 ml
1 ml
E ml diluent
$ndiluted 1 1B1
141
1B1 142
1B1 1B1 1B1 143 14! 14#
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Essential A level Biology Practical Skills
1. Decimal dilutions 'ach concentration is one tenth that of the preious one log 1 dilution series 7easure out the most concentrated solution with a 1F e%cess 7easure one4tenth of the olume required into a essel containing nine times as much diluting liquid 7i% thoroughly +epeat to obtain concentrations 1B1, 1B1, 1B1, etc times the original ?o calculate the actual concentration of solute multiply by the appropriate dilution factor 2. Doubling dilutions4 'ach concentration is half that of the preious one $se twice the olume required of the first, most concentrated solution 7easure out half of this olume into a essel containing the same olume of diluting liquid e.g. distilled water 7i% thoroughly +epeat for as many doubling dilutions as are required ?he concentrations obtained will be G, H, ⅛, etc times the original *otential 'rrors +emember thinking about these can help you ealuate your procedure2 •
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Contamination from syringes9 rinse between use or use new syringes for each solution to aoid carry4oer of solutions of the wrong concentration 1=ote that when transferring a range of prepared dilutions from sample pots into test tubes, you should start with the lowest concentration and, if you rinse the syringe in the ne%t concentration before dispensing it, you can use the same syringe or pipette2 5naccuracy in measuring olumes9 any slight inaccuracy will lead to compounded inaccuracies, so the most dilute solutions hae huge errors in concentration see precautions in measuring liquids aboe ;abel tubes carefully to ensure correct solutions are transferred 7i% solutions before transferring to ensure the correct amount of solent is added to the ne%t tube Page 5 of 16
Essential A level Biology Practical Skills
+ecording Data Don>t use scraps of paperA $se a table, which should hae4 ?itle +uled grid lines, '=C;"/5=( );; D)?), 5=C;$D5=( ?:' :')D5=(/ :eadings at tops of columns ?he independent ariable should be in the first column, beginning with the smallest alue :eadings should include units. D" ="? *$? $=5?/ 5= ?:' "DI "J ?:' ?);' /ame number of decimal places for each measurement. ?he number of places should reflect the accuracy and precision of your measurement. Do not round off data alues9 this might affect subsequent analysis of your data. = take care if using the computer as it sometimes automatically changes these "bserations of results not your interpretation of themA Write these in as much detail as possible +epeated readings9 use a separate column for each ?hink about any additional columns you may need, and draw them in at the start. )dditional columns may be used to show step by step calculations e.g. olume cm3, time s, 1Btime 1Bs, rate cm 3Bs Calculated mean aerages Do not use a greater number of decimal places than you hae in the raw data Design your table to make the recording of data as straightforward as possible, to minimise the possibility of mistakes '%plain any unusual data alues in a footnote9 don>t rely on memoryA '.g. forgot to start stopwatch • • • •
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Colorimetry
?he spectrophotometer measures the absorption of radiation in the isible and u regions of the electromagnetic spectrum. ?he spectrophotometer
Rea
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Essential A level Biology Practical Skills
allows precise measurement at a particular waelength. ) colorimeter is simpler, using filters to measure broader waelength bands e.g. green, red or blue light. *rinciples of light absorption •
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?he absorption of light is e%ponentially related to the number of molecules of the absorbing solute in the solution i.e. KCL, solute concentration )bsorbance at a particular waelength is often shown as a subscript e.g. ) ## absorbance at ## nm ?he proportion of light passing through the solution is known as transmittance ? and is calculated as the ratio of the emergent and incident light intensities. 5t is usually e%pressed as a percentage ?he colorimeter has 2 scales4 o )n e%ponential scale from -ero to infinity, measuring absorbance o ) linear scale from @ 1, measuring per cent transmittance Jor most practical purposes you should use absorbance, which is linearly related to the solute concentration KCL '%tension information Absorbance (A is !i"en by:-
) log15 oB 5 $sually shown as )%, where % the waelength in nanometres )lso4 ) εl[C] Where ε = a constant for the absorbing substance absorption coefficient l the length of the light path through the absorbing solution C concentration in molBl or gBl
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Essential A level Biology Practical Skills
Calibration Cures y preparing a set of standard solutions, each containing a known amount or concentration of a substance, and then measuring the absorbance of each solution, a Mcalibration cureN or Mstandard cureN can be produced. =ote that at high concentrations, the relationships aboe do not hold true, and the straight line relationship shown may become cured. ?he line can be used to estimate concentrations of solute in a test or unknown sample. :ow to use the colorimeter 1. /witch on 2. )llow 1# minutes for the lamp to warm up and the instrument to stabilise 3. /elect the correct coloured filter 5t is best to use the filter which selects the range of waelengths most strongly absorbed by the sample because this will gie the ma%imum reading. ?he most suitable filter colour is usually the complementary colour to the solution being tested4 C";"$+ "J /";$?5"= iolet lue lue4green (reen Iellow4green Iellow "range +ed *urple4red
J5;?'+ C";"$+ Iellow4green Iellow +ed *urple iolet lue (reen4blue lue4green (reen
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Essential A level Biology Practical Skills
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5nsert a reference blank cuette Check the reading is -ero -ero if necessary )nalyse samples Check the scale is -ero at regular interals using a reference blank e.g. after eery 1 samples P. Check the reproducibility of the instrument9 measure the absorbance of a single solution seeral times during analysis. 5t should gie the same alue 5naccuracies may be due to4 5ncorrect use of cuettes i. Dirt ii. Jingerprints iii. ?est solution on the outside of cuettes Condensation on cold samples allow cold samples to equilibrate to room temperature )ir bubbles in samples tap gently to remoe 5nsufficient solution refraction of light at meniscus Cloudiness of sample decant off supernatant to test, after allowing precipitate to settle •
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7icroscopy
*roblems in light microscopy and possible solutions4 #o ima!e$ "ery dark ima!e microscope not switched on ob6ectie nosepiece not clicked into place oer a lens lamp failure • • •
%ma!e blurred and cannot be &ocused dirty ob6ectie dirty slide slide upside down slide not completely flat on stage fine focus at end of trael • • • • •
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Essential A level Biology Practical Skills
'ust and dirt in &ield o& "iew eyepiece lens dirty ob6ectie lens dirty slide dirty dirty on lamp glass or upper condenser lens /etting up and using the light microscope • • • •
1. /elect low power lens. 7ake sure the lens clicks into position. 2. '%amine prepared slide without the microscope and note position, colour and rough si-e of specimen. 3. *lace slide on stage, coerslip uppermost, iewing it from the side. *osition it with stage ad6ustment controls so that the specimen is lit up. !. Jocus using first the course and then the fine focusing controls. $se both hands to alter the focusing controls9 this helps keep the controls working properly and not going out of alignment. =ote ?he image will be reersed and upside down when seen by iewing the slide directly. #. Jor higher magnifications, swing in the releant ob6ectie lens carefully checking there is space for it. )d6ust the focus using the fine control only. 5f the ob6ect is in the centre of the field of iew with %1 ob6ectie, it should remain in iew with the %! ob6ectie. &. When you hae finished using the microscope, ?urn the ob6ectie lens back to %1 ;ower the stage +emoe the last slide and return to correct section in tray Clean the stage if necessary Check eyepiece lenses and ob6ectie lenses are clean $nplug cable and store tidily +eplace dust coer O. (eneral care of the microscope4 =eer force any of the controls =eer touch any of the glass surfaces with anything other than clean, dry lens tissue When moing the microscope, hold the stand aboe the stage with one hand and rest the base of the stand on your other hand )lways keep the microscope ertical or the eyepiece may fall out Do not touch lenses with your fingers • • • • • • •
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Essential A level Biology Practical Skills • •
Do not allow any solent to come in contact with a lens Do not wear mascara when using the microscope9 it marks the eyepieceA
'stablishing /cale 7agnification used to obsere a specimen ob6ectie magnification % eyepiece magnification. :oweer, this is fairly meaningless when drawing any image, which could be drawn at any si-e. Jor this reason, it is essential to add a scale to your drawing. Iou can *roide a bar of defined length e.g. 1 Qm or (ie an estimated si-e of the ob6ect • •
7ethods of estimating si-e 1. Compare the si-e of the image to the diameter of the field of iew4 a. Jocus on the millimetre scale of a transparent ruler, using the lowest power ob6ectie b. 'stimate the diameter of this field directly c. $se the information to work out the field diameters of higher powers e.g. if the field at %! is ! mm, at a magnification of %1 will be !B1 % ! mm 1.& mm 2. (reater accuracy can be obtained if an eyepiece graticule micrometer is used4 a. (raticule carries a fine scale and fits inside an eyepiece lens b. (raticule is calibrated using a stage micrometer i.e. a slide with a fine scale in it Iou will do this yourself2. c. "nce you calibrate your eyepiece graticule for each ob6ectie lens used, you can use it to measure ob6ects d. 5n the e%ample below, the scale reading is multiplied by 2.&# Qm to gie the alue in micrometres. e. )oid putting too many significant figures in any estimates of dimensions9 there may be quite large errors inoled Page 11 of 16
Essential A level Biology Practical Skills
ecordin! by biolo!ical drawin!s
?he making of accurate drawings is an essential skill for )danced leel biology students. /tudents are required to make drawings of e%ternal features of whole specimens, parts of specimens, dissections and prepared slides. Drawings must be done on white )a)er usin! shar) )encil. /tudents should note the following points 1. ) drawing should be genuine and accurate record of what has been seen. Do not copy diagrams from books with little resemblance to the specimen, dissection or prepared slide under inestigation. 2. Jor low power drawings, only the outline of the structures or tissues needs to be drawn. 5ndiidual cells are required only for high power drawings. 3. Draw with bold, dark, smooth lines. /hading and the use of crayons are not encouraged. !. )oid making drawings on both sides of a paper. #. 5n making a drawing, first decide what you want to show. ?hen plan your drawing to fit on the page. 5t is important that the arious parts of the structures are drawn in proportion. &. Drawings should be large, neat and tidy. O. )ll releant structures should be fully and clearly labelled usin! )encil* 'ach label should be connected to the appropriate part of the drawing by a clear labelling line. ?he labelling lines should run as hori-ontal as possible and should not intersect with one another. Do not label too close to the drawings, and do not write on the drawing itself. Distribute the labels well around the drawing so that the labelling lines can be kept reasonably short. P. 'ery drawing should include a title, the magnification power, and the plan of iew of the specimen such as transerse section, longitudinal section. E. 7ake additional drawings if important details are too small to be shown in a low power drawing. ?his can be done by making a simple drawing of the main features, and other drawings on details of small parts only. Jor e%ample, the recording of a transerse section of a stem should include a low power plan of the section and high power drawings of different cells types as required showing the detail of the cells. Page 12 of 16
Essential A level Biology Practical Skills
1. 5f suffices to show the internal structural details in a small representatie sector of the specimen which shows repetitie features. 11. 5t is sometimes appropriate to make short succinct notes close to the labels. /uch annotated drawings are particularly aluable as they combine a record of structures with related functions andBor biological interests. Iou may want to label an organelle to describe staining e.g. nucleus darkly stained9 starch grain blue4black 'rawin! +ra)hs •
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(raphs gie a isual impression of the content and meaning of your results. ?ables proide an accurate numerical record of data alues. (raphs should4 o include all material necessary to coney the appropriate message without reference to the te%t When drawing graphs4 o Collect all data alues to be plotted o Consider whether a graph is the best way to present the data o Choose a concise e%planatory title to establish the content o Consider the layout and scale of the a%es carefully o $se the % a%is for the independent ariable o $se the y a%is for the dependent ariable o When neither ariable is determined by the other, or where ariables are interdependent, the a%es may be plotted either way round o 'ach a%is should hae a descriptie label showing what is represented and including units of measurement, separated by B or written in brackets. o 'ach a%is must hae an appropriate marked scale, showing the location of all numbers used. Jill the aailable space on the paper o 5f scale breaks are used, show them clearly o Choose the symbols for each set of data points, if plotting more than one set of data o 5nclude a key to symbols of different data sets if necessary o ?o plot ery large or small numbers, the plotted alues may be measured numbers multiplied by a power of ten e.g. 1 43 % population of bacteria B mm 3 o Draw a trend line for each set of points Cure< Page 13 of 16
Essential A level Biology Practical Skills
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/traight line< *oints 6oined with a ruler< ?his is really only alid if there is -ero error. i.e. all the plotted points are precise and accurate
)lways draw the simplest line that fits the data reasonably well and is biologically reasonable. ?ry to aoid the need to e%trapolate plotted cures by better e%perimental design. 1?his could be a point for ealuation of an e%periment2. =eer allow a computer programme to dictate what a graph looks like9 make sure you can alter scales, labels, a%es etc and make appropriate selections. Draw cures freehand if necessary.
%nter)retin! +ra)hs , Analysis and "aluation
1. Describe the relationship between the ariables, quoting data. 2. '%plain what the relationship means with reference to the biological principles inoled. 3. Consider the content a. What was the aim B hypothesis of the inestigation< b. Why were the obserations made< !. Consider what kind of graph is presented9 is it an appropriate choice for the data< #. ;ook carefully at the scale of each a%is. a. What is the starting point< b. What is the highest alue< c. Do the alues start at -ero< ) non4-ero a%is emphasi-es the differences in measurements by reducing the range of alues coered by the a%is. Page 14 of 16
Essential A level Biology Practical Skills
&. '%amine symbols and trend lines9 e.g. if 2 conditions hae been obsered while a ariable is altered, when do they differ from each other9 by how much and for how long< O. 'aluate errors4 a. ;ook for ariability in the data9 hae anomalies been recogni-ed< :ae they been included in the trend line< 5s it reasonable to ignore such points<
b. 5f mean alues are presented, the underlying errors could be large
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Essential A level Biology Practical Skills
c. :as a graph been e%trapolated correctly< ?here can be no guarantee that relationships will hold under new conditions, so the e%trapolation may be inaccurate.
d. 5s the trend line appropriate< Would a cure B straight line be more appropriate< Consider the theoretical alidity of the line. ) straight line implies one factor aries in direct relationship to another9 the true situation may be more comple% e.g. an e%ponential relationship could be more correct.
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