FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1INTRODUCTIONRevised practicals for mandatory unitsThis pack contains revised practicals for the mandatory units as detailedbelow. These practicals were first published in the HSDU support packBiology (Advanced Higher) Practical Activities, 7133, summer 2000.• Staining a root tip and calculating its mitotic indexThe concentration of disodium hydrogen phosphate (0.2 M) has beenadded to the instructions in the technical guide for the citrate/phosphate buffer used to dissolve toluidine blue.• Gel electrophoresis of DNA treated with restriction enzymesThis practical based on the NCBE Plant DNA Investigation Kit has hadmore detail and a new procedure for staining DNA added.• Isolating and examining cysts of potato cyst nematodesThe text of this practical has had some minor amendments.New practical for mandatory unitA new practical for the Cell and Molecular Biology unit, The effect ofcompetitive and non-competitive inhibitors on the enzyme β-galactosidase has been included.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2INTRODUCTIONExperimental workOne report of an experimental activity is required as evidence for theassessment of Outcome 3 in each unit. The choice of experiment is notprescribed in the unit specification and so Centres can select from theactivities included in the support materials, adapt them for individualuse, or use existing activities. The Student Activity Guides provideguidance on the amount of detail and help students might expect toreceive. The experimental activity must allow for the collection andanalysis of information to meet the performance criteria of Outcome 3.Outcome 3 performance criteria:a. The information is collected by active participation in theexperiment.b. The experimental procedures are described accurately.c. Relevant measurements and observations are recorded in anappropriate format.d. Recorded experimental information is analysed and presented inan appropriate format.e. Conclusions drawn are valid.f. The experimental procedures are evaluated with supportingargument.PurposeA range of practical activities is provided that are suitable for Outcome 3.The extension work in the teacher/lecturer guide provides ideas thatcould be developed into investigations to meet the requirements of theBiology Investigation unit.Any hazards associated with the experiments have been identified andsuitable control measures included in the support material as a result ofrisk assessment.StructureTeacher/lecturer guideThis indicates whether the experimental activity can be used to provideevidence for Outcome 3 or for other purposes. A section onbackground information includes the biology associated with theexperiment where necessary and any prior knowledge or skills studentswill require before undertaking the activity. Advice on classroommanagement for the teacher/lecturer will include advice on organising
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3INTRODUCTIONstudent groups, pooling results, time required and the supply ofmaterials to students. There will also be advice on possible extensionand follow-up activities that could be developed into ideas forinvestigations.Technical guideThis provides a list of materials required for each activity, includingsources and suppliers for items not generally available from majorsuppliers. There is advice on the preparation of materials and riskassessments. The supply of materials to students should allow for adegree of planning and organising of experimental work. This does notmean planning and designing in the sense of an investigation as oftenthe student will be presented with an experimental procedure. Rather itshould allow the student to plan how he or she will lay out equipmentand materials in preparation for carrying out the experimental activityand planning the execution of the experimental procedures.Preparing for the activityThis section is designed to make students think actively about theirexperimental work and to plan and organise its execution. To that endit includes an analysis of the activity which poses questions about theexperimental design. Students, although presented with experimentalprocedures to follow, are expected to plan and organise carrying outthe experimental work. In practical terms this will involve readingthrough the procedure, identifying and collecting the materials theyrequire and organising themselves to carry out the procedures andrecord results either individually or as a group. For some experimentalactivities ‘Preparing for the activity’ has been customised by addingevaluation questions which will assist students in considering issueswhich could be addressed in the experimental report.This section presents a number of options for teachers and lecturers inteaching experimental work. Students could be led through the stagesin preparing for the activity by their teacher/lecturer or it could bepresented to students as an individual or group activity. Alternativelythe different stages in preparing for the activity could be presented as amixture of these approaches as teachers and lecturers considerappropriate for their students. Also different experimental activitiesmay lend themselves to different approaches, or as students’ skillsdevelop the approach may be changed to suit their experience.A general section ‘Preparing for the activity’ is included as Appendix 1.This should be used for each practical activity unless there arecustomised questions on evaluation in which case a ‘Preparing for the
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)4INTRODUCTIONactivity’ section appears in the support material for that particularactivity.Student activity guideThis includes an introduction, which provides background informationfor the student on the biology of the activity or any other informationrequired. The experimental procedures for students are described inthe equipment and materials section and the instructions. Theinstructions take the students through the steps required for the activityas well as providing limited advice on the recording, analysis andpresentation of data.Conditions required for practical work for Outcome 3Arrangements documentation and Subject Guides refer to assessmentbeing carried out under controlled conditions to ensure reliability andcredibility. For the purposes of internal assessment, this means thatassessment evidence should be compiled under supervision to ensurethat it is the students’ own work.It must be emphasised that the assessment for this outcome is not aspecial assessment event but part of the ongoing learning and teachingprocess. The experimental activity is likely to be performed by a smallgroup of students together. After collection of the experimentalinformation each student must complete a report individually undersupervision. A written report should be provided for evidence wherecircumstances make that possible. For students with special needs forwhom written evidence is not appropriate alternative forms of reportcan be used.For Outcome 3 there is no specified time limit, but practical constraints,such as the length of a class period, are likely to play a part. It isappropriate to support students in producing a report to meet theperformance criteria. Thus redrafting of reports after necessarysupportive criticism is to be encouraged as part of the learning andteaching process and to produce the evidence for assessment.Redrafting should focus on the performance criteria concerned and, as ageneral rule, should be offered on a maximum of two occasionsfollowing further work by the student on the areas of difficulty.Report writingStudents should receive an ‘Advice to Candidates’ page (Appendix 2)which they can refer to during the experiment and the writing of thereport to aid clarity and ensure completeness of their report. This gives
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5INTRODUCTIONadvice on structuring the report under specific headings making a blankreport booklet unnecessary. In some experiments where only one ofthe items listed in the conclusion or evaluation is likely to be requiredthis can be indicated to the students.Marking reportsThe ‘Outcome 3: Teacher/Lecturer Guide’ in Appendix 3 summarises theperformance criteria together with suggested items which might aid theprofessional judgement of the assessor. It is important to consider eachindividual experiment and how the specific advice given in the Teacher/lecturer guide for the experimental activity relates to the suggestions toaid professional judgement. Centres may wish to produce customiseddepartmental marking schemes for the particular practical activities theyuse to provide evidence of Outcome 3. The advice on marking reportsfor Outcome 3 at Higher and Int 2 contained in the support materialMarking Advice for Assessing Outcome 3 (Int 2 and H), 5722, publishedAugust 1999, applies equally to Advanced Higher Biology.The final decision on achievement must be on the basis of theperformance criteria. Although poor grammar, poor sentenceconstruction and bad spelling would be drawn to the student’sattention, these aspects are not in any of the performance criteria.Definitive guidance on the assessment of students’ reports forOutcome 3 is to be found in National Assessment Bank materials.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 7CELL AND MOLECULAR BIOLOGYACTIVITY 1Unit: Cell and Molecular Biology (AH): Structure, function andgrowth of prokaryotic and eukaryotic cellsTitle: Staining a root tip and calculating its mitotic indexTeacher/lecturer guideType and purpose of activityThis experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the process of mitosis• develop problem solving skills and in particular Outcome 2performance criteria:(b) information is accurately processed, using calculations whereappropriate(d) experimental procedures are planned, designed and evaluatedappropriately.Background informationIn this activity students will prepare and stain root tips. To achieve anOutcome 3 students must either have two different sources of root tipsor stain one type of root tip with two different stains. A comparisonbetween either the root types or the stains will then be possible.Two recommended sources of roots are garlic and hyacinth. The garliccloves, bought normally for cooking purposes, will produce roots at anytime of year. Hyacinth bulbs can be bought at Garden Centres duringautumn and winter. Both garlic cloves and hyacinth bulbs will produceample roots for the experiment.Suitable stains for studying the stages of mitosis in root tips arelactopropionic orcein and toluidene blue.The mitotic index is the fraction of cells in a microscope field whichcontain condensed chromosomes. This index will be calculated for eachslide prepared.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)8CELL AND MOLECULAR BIOLOGYPreparation of the plant materials and the stains is covered in theTechnical Guide.To make this activity non-seasonal, it is possible to ‘fix’ the root tipswhen available and then store them until required. Fixing of root tips isonly covered in the Technical Guide.Classroom managementStudents are asked to mark the root tip one or two days prior tostaining the root tips. This will enable them to link rate of growth withmitotic index.Microscopic examination of the slides:Students should examine several slides and calculate the mitotic indexfor each one. Prepared slides could also be available.Supply of materialsIn order to satisfy the core skill in problem solving, students will berequired to identify and obtain resources required for themselves.Further advice on supply of material is given in the Technical Guide.Advice on marking Outcome 3 reportSpecific advice for performance criteria b–fPC b: a description of the preparation of the root tip(s) and themethod(s) of staining should be included.PC c: drawings or a description of some of the cells showing thedifferent stages of mitosis; the magnification used should also benoted.PC d: a table of results recording:(i) the number of cells containing condensed chromosomesin a particular field(ii) the total number of cells in the field(iii) the mitotic index for the field.The results should include at least two different microscopefields for each situation (i.e. two for each type of root tip or twofor each stain used).
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 9CELL AND MOLECULAR BIOLOGYPC e: either a conclusion is made about the rate of mitosis in thedifferent types of root tips (the higher the mitotic index thegreater the rate of mitosis) OR a conclusion is made about theefficiency of each stain for detecting condensed chromosomes.PC f: evaluation points include:• the length of time the root tips were left in the acid: if tooshort a time, maceration will be difficult; if too long a time thetip will disintegrate when being handled• the amount of cells unstained due to insufficient time in acid,poor maceration or poor uptake of stain• how efficient the stain is, e.g. the lactopropionic orceinusually gives better definition of chromosomes while thetoluidine blue is stronger in colour• the condition of the roots and their rate of growth prior tousing them for the experiment.Extension workTry to vary the mitotic index of the plant tissue, e.g. cutting the root tipsand keeping them at 0°C for 24 hours may increase the mitotic index.The experimental method can be varied, e.g. varying the temperature orconcentration of acid; varying the time the root tip is in the acid;squashing the root tip with a coverslip instead of macerating; varying theage of the root used; preparing the stains differently (e.g. differentdilutions, different pHs); heating the lactopropionic orcein slide gently;investigating a possible link between rate of growth of root and mitoticindex.AcknowledgementsInformation and advice from Dr Kwiton Jong, Royal Botanic Garden,Edinburgh, is gratefully acknowledged. Information was also receivedfrom Ashby Merson-Davies, Sevenoaks School, Kent.This experiment was produced by the SAPS Biotechnology ScotlandProject. Funding for the project was provided by SAPS, Unilever andThe Scottish Office. Support was also provided by Edinburgh University,Quest International, the Scottish CCC, the Higher Still DevelopmentUnit and the Scottish Schools Equipment Research Centre (SSERC).
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)1 0CELL AND MOLECULAR BIOLOGYTechnical guideThe class will be varying either plant material or stain for this activity.The list of materials required will vary depending on this decision.Materials requiredMaterials required by each student/group:gloves and eye protectioncompound microscope (×100 – ×400 magnification)small beaker of 1 M hydrochloric acid (2 will be required if plant materialis being investigated)small beaker of water and droppermicroscope slidescoverslipsfine forcepsdissecting needlescissorssoft tissue paperrulerfine threaddropping bottle of lactopropionic orcein and/or (see below) droppingbottle of toluidine bluegarlic clove with suitable roots and/or (see below) hyacinth bulb withsuitable rootsMaterials to be shared:waterbath at 60°Cmarker pentimerdropping bottle of 50% glyceroldropping bottle of 70% ethanollens tissuePreparation of materialsIf plant material is to be varied prepare both plant types below. If stainis to be varied prepare just any one of the plant types.To prepare hyacinth bulb roots: Place the bulb in a suitably sizedcontainer with water so that the root end is just in contact with thewater. It is best to change the water daily if possible. Roots of a suitablelength (2–6 cm) will be available within a week and perhaps sooner.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 1CELL AND MOLECULAR BIOLOGYHyacinth bulbs can cause allergies. Wear gloves if handling the bulbsregularly.To prepare garlic clove roots: Carefully peel the clove and place it in asuitably sized container with water, e.g. test tube/boiling tube so that theroot end is just immersed in the water. It is best to change the waterevery 2–3 days. Roots of a suitable length (2–6 cm) will be available after2–4 days).If stain is to be varied prepare both stains, as detailed next. If plantmaterial is to be varied prepare just any one of the stains.Wear gloves and eye protection when handling the stains.Lactopropionic orcein should be prepared in a fume cupboard or well-ventilated room. Dilute it to a 45% solution by volume with distilledwater.Toluidine blue is harmful if swallowed. Prepare a 0.5% solution in acitrate/phosphate buffer at pH4 (20 cm30.1 M citric acid + 10 cm30.2 Mdisodium hydrogen phosphate + 8 cm3distilled water).Fixing the rootsThis stage is required only if suitable roots are available but they are tobe stained at a later date.Mix 6 cm3absolute alcohol with 2 cm3glacial acetic acid in a fumecupboard. This mixture is called Farmer’s fluid and must be freshlyprepared. Once added to the Farmer’s fluid, the root tips can be storedfor many months in a refrigerator.Supply of materialsIt is not appropriate to provide all equipment and materials in, forexample, a tray system for each student/group. Equipment andmaterials should be supplied in a way that students have to identify andobtain resources. Normal laboratory apparatus should not be madeavailable in kits but should generally be available in the laboratory. Trayscould be provided containing one type of specialist equipment ormaterials.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)1 2CELL AND MOLECULAR BIOLOGYPreparing for the activityRead through the Student Activity Guide and consider the followingquestions.Analysis of activityWhat is the aim of the activity?Do you know if you are using two types of roots OR two types of stain?What measurements are you going to make?What safety measures are you required to take?As a class, decide what a ‘nucleus’ should look like for it to be composedof condensed chromosomes.In your group, decide how the activity will be managed by allocatingtasks to each member. For Outcome 3 it is important that you play anactive part in setting up the experiment and in collecting results.Recording of dataPrepare a table to record your results. You should use a ruler andappropriate headings.EvaluationIf varying plant material, was rate of growth of the two roots similar? Ifnot, is there a link between mitotic index and rate of growth?If varying stain, was there a difference in the ability of the root cells toabsorb the stains? Were they absorbed too much/insufficiently?Does the mitotic index vary much between different results? Accountfor these differences, if possible.Was the treatment in acid (step 4) sufficient to allow for both easyhandling of the root tip and easy maceration? (Insufficient acidtreatment results in easy handling but difficult maceration; too severeacid treatment results in difficult handling but easy maceration.)
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 3CELL AND MOLECULAR BIOLOGYStudent activity guideIntroductionYou are going to stain root tips and examine them for signs of cellsdividing by mitosis. The chromosomes inside the nuclei of such cellscondense and become visible. You should know what condensedchromosomes look like and how they move about inside a cell whenundergoing mitosis.Equipment and materialsMaterials required by each student/group:gloves and eye protectioncompound microscope (×100 – ×400 magnification)small beaker of 1 M hydrochloric acid (2 will be required if plant materialis being investigated)small beaker of water and droppermicroscope slidescoverslipsfine forcepsdissecting needlescissorssoft tissue paperrulerfine threaddropping bottle of lactopropionic orcein and/or (see below) droppingbottle of toluidine bluegarlic clove with suitable roots and/or (see below) hyacinth bulb withsuitable rootsMaterials to be shared:waterbath at 60°Cmarker pentimerdropping bottle of 50% glyceroldropping bottle of 70% ethanollens tissueWear gloves and eye protection whilst carrying out this experiment.Avoid skin contact with the stain(s) and avoid breathing in the fumes ofthe stain, lactopropionic orcein, if used.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)1 4CELL AND MOLECULAR BIOLOGYInstructionsEither two types of roots or two different stains will have beenprepared. Find out what is available.1. One or two days before staining the root tips, remove the plantmaterial carefully from the water and blot dry gently. Use apermanent marker pen to mark a small dot about 2 mm from theend of some root tips. Replace the plant carefully in the water.2. After one to two days, remove the plant material and use thethread and ruler to measure how much the root tips have grownsince marked.3. Preheat about 10 cm3of 1 M hydrochloric acid in a small beaker to60°C using a waterbath. Meanwhile, use a lens tissue and alcoholto clean microscope slides and coverslips.4. Using scissors, remove the last 2 mm from several young vigorouslygrowing root tips. Place them in the preheated acid and return tothe waterbath for 4–5 minutes.5. Gently transfer each root tip to a clean microscope slide containinga large drop of water.6. Gently blot dry with a piece of soft tissue.7. Using a dissection needle, thoroughly macerate the root tip andspread over an area equivalent to the size of a 5p coin.8. You are now ready to apply the stain.If using toluidine blue – Add one drop to the macerated root tipand immediately cover with a coverslip, invert the slide and blotfirmly several times on a wad of tissues.If using lactopropionic orcein – Add one drop to the maceratedroot tip and leave for 3–4 minutes. To speed up absorption of thestain, warm the slide gently by holding it 30–40cm above a yellowBunsen flame (if your hand becomes uncomfortable you areheating the slide too much). Cover with a coverslip, invert theslide and blot firmly several times on a wad of tissues.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 5CELL AND MOLECULAR BIOLOGY9. View under a microscope, ×40 – ×100 magnification initially. Scanthe slide to locate the region of mitosis.10. View this area at a higher magnification (×400 should be sufficient)and count:(i) the total number of cells in the microscope field(ii) the number of cells with condensed chromosomes which aregoing through any of the four stages of mitosis. You will have todecide where your cut-off point is when considering if cells inprophase and telophase contain condensed chromosomes (consulttextbooks).11. Repeat steps 9 and 10 for the various microscope slides prepared.If you want to prevent the slides from drying out, mount them in50% glycerol.12. Calculate the mitotic index for each slide examined (the mitoticindex is the fraction or percentage of cells containing condensedchromosomes).13. Draw a table with suitable headings summarising your results.14. Compare your results with other groups.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 7CELL AND MOLECULAR BIOLOGYACTIVITY 4Unit: Cell and Molecular Biology (AH): Applications of DNAtechnologyTitle: Gel electrophoresis of DNA treated with restrictionenzymesTeacher/lecturer guideType and purpose of activityThis experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of cutting DNA withrestriction enzymes• develop problem solving skills and in particular Outcome 2performance criteria:(c) conclusions drawn are valid and explanations given aresupported by evidence(d) experimental procedures are planned, designed and evaluatedappropriately.Background informationThis experiment is done with the help of the Plant DNA Investigation kitobtained from the National Centre for Biotechnology Education(NCBE), University of Reading, Whiteknights, PO Box 228, Reading RG66AJ. Tel: 0118 987 3743 Fax: 0118 975 0140. Cost £130.00 (2000prices). SAPS offers sponsorship towards the initial cost of a kitproviding that a teacher from the school has attended a SAPS DNAworkshop. Contact SAPS at Edinburgh University (tel: 0131 650 7124) orat Head Office (tel: 01223 507168) to obtain the appropriate form.Refills and individual items can also be obtained from NCBE. StudentGuides and a Technical Guide are supplied with the kit and these supplya great deal of relevant background information.In this experiment it is assumed that a 4-tooth gel comb is used toprovide 4 wells in each gel. If using a 6-tooth gel comb the wells holdless DNA.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)1 8CELL AND MOLECULAR BIOLOGYIn the experiment, a batch of DNA is digested by two differentrestriction enzymes. Due to inappropriate buffer concentrations, theactivity of the second enzyme will be reduced. However, evidence of itsactivity should still be apparent.Classroom managementFollowing these instructions, this experiment requires three separatedays to be completed.Day 1 – Practising with the microsyringe and digesting the DNA requires30–40 minutes followed by a 40 minute incubation at 37°C. After theincubation the small tubes should be stored in the freezer until the nextday.Day 2 – Separating the DNA fragments requires about 30 minutes to setup. Ensure the gels are loaded close to the electricity supply so they donot have to be moved once loaded. As long as the electric current hasbeen applied long enough for the DNA to have moved out of the wells(40–50 minutes at the lowest voltage) the electricity can be switched offand on as required (however, when switched off, loading dye will diffuseout of the gel making it difficult to see how far the DNA fragments havetravelled).Day 3 – Staining the gels requires only 5–10 minutes but the gel can takeanother 15–20 minutes to identify any visible bands and measure thedistance each band has travelled.The following table is a guide to suitable power supplies, voltages andtotal lengths of time to apply voltage to obtain good separation of DNAfragments.Students hands should be dry when carrying out the electophoresis.Type of power supply Maximum safe voltage Time to runbattery (4 × 9 V) 36 V about 2 hours*regulated power pack 30 V about 2.5 hoursunregulated power pack 16 V 5–6 hours*regulated power packs can be identified either by labels on theapparatus or from their accompanying technical information.Supply of materialsIn order to satisfy the core skill in problem solving, students will berequired to identify and obtain resources required for themselves.Further advice on supply of material is given in the Technical Guide.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 9CELL AND MOLECULAR BIOLOGYAdvice on marking Outcome 3 reportSpecific advice for performance criteria b–fPC b: an outline of procedure being carried out each day, e.g. Day 1 –each restriction enzyme cutting up the DNA at specific points;Day 2 – the electricity causing the DNA fragments to migratethrough the gel, the rate of movement being linked to the sizeof the fragment; Day 3 – the DNA is stained and the number ofbase pairs in any visible band identified by referring to the tablesupplied.PC c: a table of results with appropriate headings and units showingthe size of each visible DNA fragment and the distance it hastravelled.PC d: a graph of the results. It is probably best with the size of DNAfragment (number of base pairs) on the x-axis and the distancetravelled (mm) on the y-axis. (If the log of the size of DNAfragment is plotted against the distance travelled a straight lineshould be formed.)PC e: a conclusion stating that the smaller the DNA fragment thefurther it will travel; however, the relationship is not linear, e.g.a small fragment half the size of another fragment will travelmore than twice the distance of the larger fragment.(Alternatively, the conclusion could state that there is a linearrelationship between the log of the size of DNA fragment andthe distances moved through the gel.)PC f: evaluation points include:• was the DNA mixed enough each time it was transferred? Iftoo much DNA is in a well ‘streaking’ of the bands will occur;too little DNA in a well will result in faint bands.• was the electricity switched on the correct length of time andan appropriate voltage used? DNA bands should be spacedout over the entire gel; appropriate voltage is 1–5 volts percentimetre (the distance between the two electrodes).• corrosion may occur at the anode; despite this, theelectrophoresis should not be affected.• if the gel is blank then either the DNA has not beenadequately rehydrated or the stain has not been left incontact with the gel for long enough.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2 0CELL AND MOLECULAR BIOLOGY• why are the smaller DNA fragments not visible? What sizemust the fragments in your gel be before they are visible?• why have some fragments not separated sufficiently to beseen as separate bands?ReferencesInvestigating Plant DNA – Student Guide and Technical Guide. Thesebooklets accompany the DNA kit available from NCBE.Micklos, D. and Freyer, G. (1990), DNA Science. A first course inrecombinant DNA technology, Cold Spring Harbour Laboratory Press/Carolina Biological Supply Company.Miller, M. B. (1993), ‘DNA technology in schools: a straightforwardapproach’, Biotechnology Education, 4(1), 15–21.Miller, M. B. (1994), ‘Practical DNA technology in school’, Journal ofBiological Education, 28(3) 203–211.Miller, M. B. and Russell, G. A. (1996), ‘Practical DNA technology inschool – 2: Computer analysis of bacteriophage lambda base sequence’,Journal of Biological Education, 30(3) 176–183.http://www.ncbe.reading.ac.ukAcknowledgementThis experiment was produced by the SAPS Biotechnology ScotlandProject. Funding for the project was provided by SAPS, Unilever andThe Scottish Office. Support was also provided by Edinburgh University,Quest International, the Scottish CCC, the Higher Still DevelopmentUnit and SSERC.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 2 1CELL AND MOLECULAR BIOLOGYTechnical guideMaterials requiredMaterials required by each student/group:Day 1 – 2 pink tubes containing the restriction enzyme EcoR12 green tubes containing the restriction enzyme Hind1111 yellow tube (empty)1 white tube of DNA suspension1 microsyringe and 6 tips1 float1 vial of loading dye1 piece of parafilm1 marker penDay 2 – electrical supply (see Teacher/Lecturer Guide)2 electric wires with crocodile clipsenzyme tubes in the float from the previous lessonvial of loading dyegel in a plastic tank with comb, covered in buffer solutionmicrosyringe and 4 tipspiece of black card2 pieces of carbon fibre tissueDay 3 – tank containing your gel from previous lessonstain (10 cm3)gloveseye protectionMaterials to be shared:Day 1 – waterbath at 37°CDay 2 – bottle of TBE bufferPreparation of materialsPreparation of materials supplied by the kitRehydrating the DNA – The λ DNA in the narrow white tubes provided inthe Plant DNA kit must be rehydrated with distilled water shortly beforethe experiment is carried out. Follow the instructions on page 10 of theStudent Guide provided with the kit. One tube of DNA is required pergroup of students.Preparing the agarose gel – If necessary, this can be done a few daysbefore the experiment is carried out. Follow the instructions on page12 of the Student Guide. One gel is required per group of students.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2 2CELL AND MOLECULAR BIOLOGYTwo pieces of carbon fibre electrode tissue (approximately 42 mm ×22 mm) are required per group. Wear gloves when handling the carbonfibre tissue.Dilute 1 volume of the TBE buffer concentrate with 9 volumes ofdistilled water. About 35 cm3will be required per group (11–12 cm3todissolve the agarose and form the gel and the rest to cover the gel onceit is set). The liquid can be reused for 3–4 ‘runs’ after which it should bediscarded.Dilute the concentrated stain for DNA with an equal volume of distilledwater. About 10 cm3of stain is required per group. This diluted staincan also be reused several times. Wear gloves and eye protection whenhandling the stain.Recipes for the various buffers and dyes used in the experiment aregiven in the Technical Guide supplied with the kit.Preparation of materials not supplied by the kitMaking a float – Make 4–5 holes in a plastic petri dish lid or base using asmall hot rod. The holes should be about 8 mm in diameter. This willallow the pointed end of the enzyme microtubes through but will holdtheir top end. Alternatively, the holes can be made in a thin piece offoam such as a camping mat.Pieces of Parafilm (about 5 cm × 5 cm) are required for the microsyringeexercise. However, any non-absorbent paper such as benchcoat will besuitable.9 volt PP3 batteries can be obtained very reasonably (70p each – 2000prices) from Middlesex University Services Ltd, (Teaching Resources),Trent Park, Bramley Road, London N14 4YZ.Tel: 0208 4470342 Fax: 0208 447 0340.Supply of materialsIt is not appropriate to provide all equipment and materials in, forexample, a tray system for each student/group. Equipment andmaterials should be supplied in a way that students have to identify andobtain resources. Normal laboratory apparatus should not be madeavailable in kits but should generally be available in the laboratory. Trayscould be provided containing one type of specialist equipment ormaterials.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 2 3CELL AND MOLECULAR BIOLOGYDisposal of materialsAll microtubes and gels can be safely disposed of in the bin. Buffer,loading dye and stain can be diluted and washed down the drain. Afuller account of safety is covered in the Technical Guide accompanyingthe kit.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2 4CELL AND MOLECULAR BIOLOGYPreparing for the activityRead through the Student Activity Guide and consider the followingquestions.Analysis of activityWhat is the aim of the activity?What measurements are you going to make?Are you familiar with how the restriction enzymes act on DNA?Are you aware of what is happening during electrophoresis?Getting organised for experimental workWhat safety measures are you required to take?Are you familiar with the microsyringe and how to deliver a set volumeusing it?Recording of dataPrepare a table with suitable headings and units to record the number ofbase pairs in each identified DNA fragment and the distance it hastravelled through the gel.EvaluationWhy are some DNA fragments not visible?Why have some DNA fragments not separated sufficiently to be seen asseparate bands?Is there evidence that the DNA was not evenly distributed in its originaltube? What can be done to prevent this?How long should the electric current be passed through the gel so thatDNA bands will be separated as much as possible?Can you account for some lanes of the gel being blank?
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 2 5CELL AND MOLECULAR BIOLOGYStudent activity guideIntroductionThis experiment uses most of the basic techniques involved in geneticfingerprinting. The DNA is digested or ‘cut up’ using restrictionenzymes. The resulting fragments of DNA are then separated into bandsusing an electric current and made visible by staining.DNA source DNA fragments DNA fragmentsof varying size separated and stainedIf the order of bases in the DNA used is different each time then theDNA fragments produced each time after digestion will also be different.Thus, DNA from different organisms (except clones) will give a uniqueresult in this experiment – hence the term genetic fingerprinting.DNA from a certain bacteriophage will be used in this experiment asonly one, short chromosome is present in the organism. This will resultin only a few different fragments being formed, thus making theirseparation into distinct bands more likely.Nuclear DNA from animals or plants consists of many largechromosomes. After digestion, a very large number of fragments areformed. If all these fragments were stained, a smear would result. Toobtain distinct bands (a fingerprint) with this complex DNA, only certainfragments are selected using probes.The simple, bacteriophage DNA is going to be digested in 3 differentways:– by mixing one sample of DNA with a restriction enzyme called EcoRI– by mixing another sample of DNA with a different restriction enzymecalled HindIII– by mixing a third sample of DNA with both of these enzymes.DNA cut withrestrictionenzymeselectriccurrent
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2 6CELL AND MOLECULAR BIOLOGYEach restriction enzyme will cut the DNA only when a certain sequenceof bases occurs, e.g. the enzyme EcoR1 cuts the DNA between bases Gand A only when the sequence GAATTC is present in the DNA. Theother restriction enzyme used cuts the DNA at a different sequence ofbases. Thus, each restriction enzyme is specific.restriction enzyme EcoRIThe number of DNA fragments formed after digestion by an enzyme willdepend on the number of times the particular sequence of bases whichthe enzyme acts on is present, e.g. the sequence GAATTC occurs 5 timesin the bacteriophage DNA used in this experiment. The DNA willtherefore be cut into six fragments when digested by the enzyme EcoRI.Equipment and materialsMaterials required by each student/group:Day 1 – 2 pink tubes containing the restriction enzyme EcoRI2 green tubes containing the restriction enzyme HindIII1 yellow tube (empty)1 white tube of DNA suspension1 microsyringe and 6 tips1 float1 vial of loading dye1 piece of Parafilm1 marker penDay 2 – electrical supply2 electric wires with crocodile clipsenzyme tubes in the float from the previous lessonDNAdoublehelixDNA cutintofragmentsGCTA G C T T A AC G A A T TGC G A CC T GGCTA G C T T A AC G A A T TGC G A CC T G
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 2 7CELL AND MOLECULAR BIOLOGYvial of loading dyegel in a plastic tank with comb, covered in buffer solutionmicrosyringe and 4 tipspiece of black card2 pieces of carbon fibre tissueDay 3 - tank containing your gel from previous lessonstain (10 cm3)gloveseye protectionMaterials to be shared:Day 1 – waterbath at 37°CDay 2 – bottle of TBE bufferInstructionsPreliminary exerciseThis experiment requires you to transfer very small volumesof liquids. A microsyringe is provided for you to do this. Thetips that fit on the end of the microsyringe have small ‘ridges’on them. When the tip is filled to the upper ridge 10 µl willbe delivered. The lower ridge is for delivering volumes of2 µl.Follow the hints below when using a microsyringe.• Before loading the microsyringe, pull the plunger out a little. Thisgives some extra air with which to expel the last drop of liquid.• When emptying the microsyringe tip, hold it vertically and at eyelevel.• To remove the last droplet from the tip, touch it against the inner wallof the container.• Do not touch the point of the microsyringe tip with your fingers.There are enzymes in sweat which may contaminate and result inunwanted digestion of DNA samples.• A tip must only be used once to prevent any cross-contaminationoccurring.← 10 µl← 2 µl
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)2 8CELL AND MOLECULAR BIOLOGYMicrosyringe exerciseYou may find this useful to become familiar with the microsyringe.i) Draw in 2 µl of dye and deposit as drop 1 on the Parafilm.ii) Repeat Step 1 until you have 5 separate drops of dye.iii) Draw in 10 µl of dye and deposit it alongside the smaller drops.iv) Now draw all five 2 µl drops into the micropipetter tip and depositthem alongside the 10 µl drop.v) Are the two drops the same size?Day 1 – Digesting the DNA1. Sit the 4 tubes containing restriction enzymes in the float on thebench.2. With a new microsyringe tip draw the DNA suspension into andout of the microsyringe tip several times. This results in the DNAbeing evenly distributed. Now transfer 20 µl of DNA to each of thetwo pink tubes containing a restriction enzyme.3. Again with a new tip, transfer 20 µl of DNA to one green tubecontaining a different restriction enzyme. Remember to mix theDNA thoroughly before transferring it.4. Again with a new tip, transfer 20 µl of DNA to an empty yellowtube. This tube will act as a control as here the DNA will beundigested.5. Cap the tubes and flick the sides of the tubes with one finger untilthe blue colour is evenly spread throughout the liquid.6. Place the float with the 4 tubes in awaterbath at 37°C (leaving the oneremaining green tube on your bench).7. After 10 minutes the restrictionenzymes will be in solution. This willallow you to transfer the entirecontents of one of the pink tubes to
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 2 9CELL AND MOLECULAR BIOLOGYthe remaining green tube again using a new tip on themicrosyringe.The DNA in this green tube will now be digested by bothrestriction enzymes. Mark the tube with a D – for double digest.8. Flick each tube several times to mix the contents. Put the fourtubes (one pink, one unmarked green, one green marked D andone yellow) in the float back into the waterbath to incubate at 37°Cfor at least another 30–40 minutes.N.B. The tubes can be left until next lesson as the restrictionenzymes will become denatured after a few hours. To preventfurther DNA breakdown, the tubes should be stored in a freezerovernight.Day 2 – Separating the DNA fragments1. If not already done, cover the gel with about 20 cm3of buffersolution (to a depth similar to that shown in the diagram below).Buffer solutions keep the pH stable and thus prevent unwantedbreakdown of unstable molecules such as DNA.2. Remove the comb gently from the gel to expose the wells.3. Ensure your tank is close to your electricity supply and place apiece of black card under it to make the wells more visible.*4. Using a new tip, draw in 2 µl of loading dye and mix thisthoroughly with the undigested DNA in the yellow tube by drawingthe mixture up and down in the tip several times.*5. Draw up all the contents of the tube into the microsyringe tip andload well 1 by emptying the syringe slowly when the end of the tipis in the buffer solution and directly above the well.N.B. The tip does not actually need to be in the well as the densedye will make the DNA solution sink.loading dyeand DNAbuffer solutiongelmicrosyringe tip
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)3 0CELL AND MOLECULAR BIOLOGY6. Repeat the last two steps marked * and load each well as follows,using a new microsyringe tip each time:Well 2 – DNA digested by restriction enzyme EcoRI (pink tube)Well 3 – DNA digested by restriction enzyme HindIII (green tube)Well 4 – DNA digested by both restriction enzymes (green tube D)7. Put a piece of carbon fibre tissue at either end of the tank.8. Connect the carbon tissue to the electricity supply using wires andcrocodile clips. Once the electricity is switched on the negativelycharged phosphates in the DNA are attracted to the positiveelectrode. So, make sure the positive electrode is furthest awayfrom the DNA in the wells.9. Check with your teacher what voltage you will be using and set upthe electricity supply accordingly. Switch on the electricity. TheTBE buffer can evaporate during electrophoresis, so periodicallycheck the depth of the buffer and top up as required (to a depthsimilar to that shown in the diagram in Step 5).As well as helping the DNA sink into the wells, the loading dye alsoallows us to judge how long the electric current should be on bymoving in front of all but the smallest DNA fragments.10. After an appropriate time (e.g. 12 hours at 9 volts; 6 hours at 18volts) switch off the electricity, disconnect the crocodile clips andremove the pieces of carbon fibre.carbon fibrewellsbuffer solution
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3 1CELL AND MOLECULAR BIOLOGYDay 3 – Staining the DNA1. Return the buffer solution covering the gel to its originalcontainer.2. Pour about 10 cm3of staining solution (Azure A) onto the surfaceof the gel and leave it for at least 4 minutes.3. Pour off the stain into a bottle labelled ‘reused stain’.4. Wash excess stain from the surface of the gel with tap water.5. Do not leave any water on the gel after rinsing. If you do the stainwill move out of the gel into the water.If the staining solution has been used on a previous occasion you mayneed to repeat the above procedure. If this is necessary allow at least 10minutes for instruction 2.Purple bands of stained DNA will appear shortly. The smaller thefragments of DNA the further it will have travelled through the gel.However, the smallest fragments will also take up less stain and maytherefore be difficult to see. Also, fragments of similar size will movesimilar distances in the gel, resulting in little separation between them.On the next page is a table showing the number and size of DNAfragments formed during the experiment. This is possible as the entirebase sequence of the DNA in the bacteriophage used has been workedout.Lanes1 2 3 4DNAbandslargestsmallestloading dyewells
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)3 2CELL AND MOLECULAR BIOLOGYLane 1 Lane 2 Lane 3 Lane 4Contents Undigested DNA digested DNA digested DNA digestedDNA by restriction by restriction by bothenzyme, enzyme, restrictionEcoRI HindIII enzymesNo. of DNAfragments formed 1 6 8 13No. of log of 48,502 4.685 21,226 4.327 23,130 4.364 21,226 4.327base fragment 7,421 3.870 9,416 3.974 5,148 3.712pairs in size 5,804 3.764 6,557 3.817 4,973 3.697each 5,643 3.752 4,361 3.640 4,268 3.630fragment 4,878 3.688 2,322 3.366 3,530 3.5483,530 3.548 2,027 3.307 2,027 3.307564 2.751 1,904 3.280125 2.097 1,584 3.2001,375 3.138947 2.976831 2.920564 2.751125 2.0976. Examine your gel and try to connect the DNA fragments listedabove with the bands that have appeared in each lane. For eachidentifiable band measure the distance it has travelled. Measurefrom the bottom of each well to the front end of each band.7. Make a table with appropriate headings and units showing thenumber of base pairs, the log of the fragment size and the distancetravelled for each band.8. Present your results as a graph with suitable scales and axeslabelled with quantities and units (put fragment size or log offragment size on the x-axis and distance travelled on the y-axis).
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3 3CELL AND MOLECULAR BIOLOGYUnit: Cell and Molecular Biology (AH): Molecular interactions incell events: CatalysisTitle: The effect of competitive and non-competitive inhibitorson the enzyme β-galactosidaseTeacher/lecturer guideType and purpose of activityThis experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the effect of competitiveand non-competitive inhibitors on enzyme activity• develop problem solving skills and in particular Outcome 2performance criteria:(c) conclusions drawn are valid and explanations given aresupported by evidence(d) experimental procedures are planned, designed and evaluatedappropriately.Background informationThe enzyme β-galactosidase catalyses the following reaction:LACTOSE GLUCOSE + GALACTOSEThe chemical ONPG (o-nitrophenyl β-D-galactopyranoside) is alsodegraded by the enzyme:ONPG ONP + GALACTOSEThe ONP produced is yellow, allowing the rate of this reaction to befollowed colorimetrically.Galactose acts as a competitive inhibitor, competing with ONPG for theactive site of the enzyme. At a sufficiently high concentration, it willinhibit the reaction by preventing ONPG making contact with the activeACTIVITY 8β-galactosidaseβ-galactosidase
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)3 4CELL AND MOLECULAR BIOLOGYsite. The enzyme, however, is still capable of activity. Thus, when theONPG concentration is increased it will eventually overcome theinhibition.Iodine solution on the other hand is a non-competitive inhibitor. Whenit combines with the enzyme the shape of the active site is alteredsufficiently to prevent the substrate combining with it. Increasingsubstrate concentration will therefore not overcome the inhibition.Classroom managementStudents can work individually or in pairs for this experiment.If there are several groups of pupils requiring to use the colorimeter, arotation system could perhaps be employed, i.e. each group could startthe reaction (by adding the enzyme) 20–30 seconds apart. Thecolorimeter would just require to be zeroed once for each ‘run’. In thisway 4–6 groups could carry out the experiment at about the same time.Estimated time: 50–60 minutes should be sufficient to collect all thedata.The enzyme solution must be kept in crushed ice. If allowed to reachroom temperature its activity will rapidly decrease.Supply of materialsIn order to satisfy the core skill in problem solving, students will berequired to identify and obtain resources required for themselves.Further advice on supply of material is given in the Technical Guide.Advice on marking Outcome 3 reportSpecific advice for peformance criteria b–fPC b: to include a description of the contents of the various cuvettesset up; preparation of the enzyme solution.PC c: a table of results for each inhibitor with appropriate headings(volume of stock ONPG solution present (cm3) and absorbance/transmission after two minutes); a table of results using the ×20diluted ONPG without inhibitor at the beginning and end of theexperiment.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3 5CELL AND MOLECULAR BIOLOGYPC d: results for each inhibitor are graphed with volume of stockONPG added on the x-axis and absorption/transmission after twominutes on the y-axis; an appropriate scale is used and axes arelabelled with units; the points are correctly plotted and lines ofbest fit are drawn.PC e: a conclusion is made as to the type of inhibitor galactose andiodine solutions are.PC f: evaluation points include:• evidence that the activity of the enzyme has remained aboutconstant throughout the duration of the experiment• suitable precautions have been taken to prevent cross-contamination• the importance of keeping the concentration of eachinhibitor constant while increasing the ONPG concentration• the suitability of the concentration of inhibitor used (did itinhibit the ×20 diluted ONPG completely?) and the range ofONPG concentrations used (did enzyme activity recover to itsinitial level when ONPG concentration was high?)• why it is more difficult to obtain complete inhibition withgalactose than with iodine solution.Extension workSubstitute galactose for glucose (the other product of the reaction) tosee if it has a similar effect on enzyme activity.Investigate the rates of reaction in the above experiment by regularlymeasuring absorbance/transmission over 5–6 minutes.Investigate the nature of the inhibition using the enzyme phosphataseand the inhibitors phosphate and iodine.The rate of reaction (V0) at low substrate concentrations can becalculated. If 1/V0is plotted against 1/[substrate] then the maximumvelocity and the Michaelis constant for the reaction can be calculated.See Hames reference on enzyme kinetics (or any good biochemistrytextbook).
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)3 6CELL AND MOLECULAR BIOLOGYReferencesAdds, Larkcom and Miller (eds.), (1996) Cell Biology and Genetics,Nelson Advanced Modular Science.Hames, B.D., Hooper, N.M. and Houghton, J.D. (1997), Instant Notes inBiochemistry, Bios Scientific.Russo, S. E. and Moothart, L. (1986), ‘Kinetic study of the enzymelactase’, Journal of Chemical Education, 63(3), 242–243.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3 7CELL AND MOLECULAR BIOLOGYTechnical guideMaterials requiredMaterials required by each student/group:6 cuvettes (or test tubes if suitable colorimeter is used)2 boiling tubesbeaker of crushed ice6 × 1 cm3droppers10 cm3syringe6 cm3ONPG stock solution (3 × 10-2M in buffer)40 cm3buffer (0.1 M potassium phosphate, pH 8)15 cm320% galactose in buffer5 cm3I2/KI solution in buffer25 cm3distilled watereye protectionglovesMaterials to be shared:colorimeter (420–440 nm filter)1 cm3dropperdistilled waterβ-galactosidase stock solutionPreparation of materialsThe buffer: 0.1 M K2HPO4adjusted to pH 8 with 0.5 M HCl. Eachstudent/group will require 80–100 cm3. About half the volume made upwill remain as plain buffer. The rest will be used to make up othersolutions. Avoid direct skin and eye contact, wear eye protection andgloves.ONPG stock solution: 3 × 10-2M in buffer. Each student/group willrequire 6 cm3. For every 10 cm3required, weigh out 0.09 g and dissolvein 10 cm3buffer. Shaking for 5–10 minutes will be required for thepowder to be completely dissolved. The ONPG stock solution is bestmade up fresh (or no more than 2 days in advance and stored in thefridge). ONPG available from Sigma Aldrich, Fancy Road, Poole, DorsetBH12 4QH. Catalogue no. N1127, 1 g for £9.70 (1999 prices).Galactose solution: 20% in buffer. Each student/group will require10–15 cm3. To make up 50 cm3, dissolve 10 g galactose in 50 cm3buffer.It dissolves readily.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)3 8CELL AND MOLECULAR BIOLOGYI2/KI solution: Each student/group will require about 5 cm3. Dissolve0.3 g iodine and 1.5 g potassium iodide in 100 cm3water to make a stocksolution (this will keep for months stored in a dark glass bottle). Take1 cm3of this stock solution and make up to 80 cm3with buffer. Thisdiluted I2/KI solution is the solution to be used by the students in theexperiment.Iodine is classified as harmful. Wear gloves when preparing thesolution.N.B. The diluted I2/KI solution must be made up immediately beforethe experiment is carried out (it will remain effective as an inhibitor for1 hour).β-galactosidase is available as ‘Lactozym’ from NCBE, University ofReading, Whiteknights, PO Box 228, Reading RG6 6AJ. Tel: 0118 9873743. Fax: 0118 975 0140. Cost £12.50 (2000 prices) for 100 cm3.Avoid direct skin and eye contact, wear eye protection and gloves.Enzyme powder can cause allergies. Do not allow any spillages to dryup. Wipe up spillages immediately and rinse cloth thoroughly withwater.For guidance on sources of colorimeters see SSERC Bulletin No. 198,Winter 1999/2000, pages 20–27.Supply of materialsIt is not appropriate to provide all equipment and materials in, forexample, a tray system for each student/group. Equipment andmaterials should be supplied in a way that students have to identify andobtain resources. Normal laboratory apparatus should not be madeavailable in kits but should generally be available in the laboratory. Trayscould be provided containing one type of specialist equipment ormaterials.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 3 9CELL AND MOLECULAR BIOLOGYPreparing for the activityRead through the Student Activity Guide and consider the followingquestions.Analysis of activityWhat is the aim of the activity?What is being varied in the activity?What variables must be kept constant?What measurements are you going to make?Why should the enzyme activity be measured without either inhibitorboth at the beginning and at the end of the experiment?Getting organised for experimental workWhat safety measures are you required to take?In your group decide how the activity will be managed by allocatingtasks to each member. For Outcome 3 it is important that you play anactive part in setting up the experiment and in collecting results.Recording of dataPrepare tables to record your group results.You should use a ruler, correct headings and appropriate units.EvaluationHas the activity of the enzyme remained about constant for the durationof the experiment?Cross-contamination will seriously affect the results. Have sufficientmeasures been taken to avoid cross-contamination?Why is it more difficult to completely inhibit the enzyme with galactosethan with iodine solution?Is the range of ONPG concentrations used suitable to show clearly if theinhibitor is competitive or non-competitive?
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)4 0CELL AND MOLECULAR BIOLOGYStudent activity guideIntroductionInhibitors are substances that reduce the activity of enzymes.When the inhibitor binds reversibly to the active site of the enzyme it isknown as a competitive inhibitor. Often a competitive inhibitor is asimilar shape to the substrate. Its association with the active site of theenzyme reduces the rate of binding between the substrate and theenzyme, thus lowering the rate of reaction. However, this type ofinhibition can be overcome by increasing the substrate concentration asthis will decrease the chances of enzyme and inhibitor binding.When a non-competitive inhibitor combines with an enzyme, theactive site may still be free. When it combines with the enzyme the shapeof the active site is altered sufficiently to prevent the substratecombining with it. Increasing substrate concentration will therefore notovercome the inhibition.In this experiment you will use the enzyme β-galactosidase. Its normalsubstrate is lactose but you will use a synthetic substrate, ONPG. Whenthe enzyme is active, it breaks down the ONPG to a yellow substance.Thus, the rate of reaction is proportional to the intensity of the yellowcolour formed.ONPG YELLOW SUBSTANCE + GALACTOSE(ONP)The reaction will firstly be carried out without an inhibitor, using a lowconcentration of substrate. An inhibitor will then be used at aconcentration that prevents this enzyme/substrate mixture fromreacting. While keeping the inhibitor concentration constant, thesubstrate concentration will be gradually increased. If the inhibition isovercome by this action, the inhibitor is competitive. If the inhibition isunaffected, the inhibitor is non-competitive.β-galactosidase
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 4 1CELL AND MOLECULAR BIOLOGYEquipment and materialsMaterials required by each student/group:6 cuvettes (or test tubes if suitable colorimeter is used)2 boiling tubesbeaker of crushed ice6 × 1 cm3droppers10 cm3syringe6 cm3ONPG stock solution (3 × 10-2M in buffer)40 cm3buffer (0.1 M potassium phosphate, pH 8)15 cm320% galactose in buffer5 cm3I2/KI solution in buffer25 cm3distilled watereye protectionglovesMaterials to be shared:colorimeter (420–440 nm filter)1 cm3dropperdistilled waterβ-galactosidase stock solutionInstructionsWear eye protection and gloves throughout this experiment to avoiddirect skin and eye contact with some of the chemicals used.1. Put 20 cm3of distilled water in a boiling tube. Surround the tubewith crushed ice and add 4 drops of β-galactosidase.This is the enzyme solution you will use throughout theexperiment. Do not allow it to reach room temperature as thiswill reduce the enzyme’s activity considerably. Ensure the stockβ-galactosidase is returned to the refrigerator as soon as possible.Enzyme powder can cause allergies. Do not allow any spillages todry up. Wipe up spillages immediately and rinse cloth thoroughlywith water.2. Mix 0.5 cm3of the stock ONPG solution with 9.5 cm3of 0.1M buffer(pH 8). Label ×20 dilution.3. Put 2 cm3of buffer and 1 cm3of this ×20 diluted ONPG solutioninto a cuvette. Mix by inverting the cuvette 2–3 times. Zero thecolorimeter with this solution.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)4 2CELL AND MOLECULAR BIOLOGY4. Add 0.5 cm3of the diluted enzyme to the cuvette. Start thestopclock and invert the cuvette 2–3 times.5. Record the absorbance/transmission two minutes after adding theenzyme. This should be between 0.3 and 0.5 absorbance units(50–32% transmission).If the absorbance is above 0.5 units, dilute the enzyme solutionwith distilled water and repeat steps 2–4 until an appropriateabsorbance is obtained after 2 minutes. If the absorbance is below0.3 units, add 1–2 drops of the stock β-galactosidase to yourdiluted enzyme.You are now going to investigate:(i) the effect of galactose (an inhibitor) on the activity of the enzyme(ii) the effect of increasing the ONPG concentration (the substrate) inthe presence of galactose.6. Mix the solutions, as shown in the following table, in differentcuvettes.cuvette no. 20% galactose ONPG stock buffer (cm3) *ONPG ×20in buffer (cm3) solution (cm3) dilution(cm3)1 2 - - 1.02 2 0.25 0.75 -3 2 0.5 0.5 -4 2 0.75 0.25 -5 2 1.0 0 -* Note: the volume of ONPG stock solution in the ×20 dilution is 0.05 cm37. Treat each cuvette in turn as follows:Invert 2–3 times, put in colorimeter and zero the instrument.(Care! If you are sharing the colorimeter with other groups, onlythe first group should zero it for each ‘run’.)Add 0.5 cm3of the diluted enzyme solution. Start the stopclockand invert cuvette 2–3 times.Take an absorbance/transmission reading 2 minutes after addingthe enzyme. Record your results in a table with suitable headings.Rinse out the cuvettes several times with water and dry.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 4 3CELL AND MOLECULAR BIOLOGYYou are now going to investigate:(i) the effect of iodine solution (another inhibitor) on the activity ofthe enzyme(ii) the effect of increasing the ONPG concentration in the presence ofthe iodine solution.Care! Iodine is harmful. Wear gloves and eye protection.8. Again, using the following table as a guide, mix the solutions indifferent cuvettes.cuvette no. I2KI solution ONPG stock buffer (cm3) *ONPG ×20(cm3) solution (cm3) dilution(cm3)1 1.0 - 1.0 1.02 1.0 0.5 1.5 -3 1.0 1.0 1.0 -9. Treat each cuvette in turn as follows:Invert 2–3 times, put in colorimeter and zero the instrument.(Care! If you are sharing the colorimeter with other groups, onlythe first group should zero it for each ‘run’.)Add 0.5 cm3of the diluted enzyme. Start the stopclock and invertcuvette 2–3 times.Take an absorbance/transmission reading 2 minutes after addingthe enzyme. Record your results in a table with suitable headings.Rinse out the cuvettes several times with water and dry.10. To ensure that enzyme activity has remained constant, repeat steps3–5. These results should be similar to the ones obtained initially.11. Present your results for both investigations as a graph with suitablescales and axes labelled with quantities and units.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 4 5ENVIRONMENTAL BIOLOGYACTIVITY 5Unit: Environmental Biology (AH): Symbiotic relationships(Parasitism)Title: Isolating and examining cysts of potato cyst nematodesTeacher/lecturer guideType and purpose of activityThis experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of parasitism and morespecifically of the relationship between potato cyst nematodes (PCN)and potato plants• develop problem solving skills and in particular Outcome 2performance criteria:(b) information is accurately processed using calculations whereappropriate(d) experimental procedures are planned, designed and evaluatedappropriately.Background informationAn outline of the life cycle, transmission and control of the potato cystnematode (PCN) is covered in the Student Activity Guide.This is a good example of parasitism to study as:(i) it affects a common and economically important food crop(ii) cysts containing the parasite remain viable for many years and canbe collected and examined at any time of year(iii) controlling PCN is expensive, complicated and an ever increasingproblem.There are two species of PCN; Globodera rostochiensis and Globoderapallida. Although both are troublesome, G. pallida is the more seriouspest and becoming increasingly difficult to control. Some varieties ofpotato are resistant to G. rostochiensis. A few varieties are partially
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)4 6ENVIRONMENTAL BIOLOGYresistant to G. pallida. Varieties susceptible to both are: Arran Comet,Desiree, Estima, King Edward, Maris Bard, Maris Peer, Pentland Dell,Record, Wilja, Golden Wonder and Kerr’s Pink. Resistant varieties to G.rostochiensis include: Cara and Maris Piper. Nadine and Sante areresistant to G. rostochiensis and partially resistant to G. pallida.Classroom managementObtaining suitable soil samples is covered in the Technical Guide. Theinitial extraction of PCN using sieves should take only 15–20 minutes.However, filtering the water/soil mixture must be completed beforeproceeding to the next stage of the experiment. The filtering will takeabout 30 minutes and, of course, longer if the water/soil mixture isfiltered a second time.Ideally the moist filter papers should be kept overnight in a humidenvironment. The cysts will then burst more readily. However, it ispossible to complete the entire experiment on the same day if necessary,although cyst bursting may be less successful.Examination of the cysts will take 30–60 minutes. The filter papers arefirst examined under a low power binocular microscope (×10 – ×20).Cysts are transferred to a microscope slide and then burst whilst viewingunder a compound microscope (×100). Identifying PCN cysts anddistinguishing between viable and non-viable PCN is covered in theStudent Activity Guide.N.B. PCN are a serious pest of a common food crop and as suchare subject to statutory control measures to limit their spread andpopulation increase. It is therefore essential that good laboratorypractice is followed at all times during this procedure. Thisincludes autoclaving all possible sources of viable cysts once theexperiment is completed. All possible precautions should also befollowed to prevent soil infected with viable cysts from beingwashed down the sink, especially if sludge from local sewagetreatment plants is spread on agricultural soil. Care must also betaken to avoid cross-contamination of samples.Supply of materialsIn order to satisfy the core skill in problem solving, students willbe required to identify and obtain resources required forthemselves. Further advice on supply of material is given in theTechnical Guide.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 4 7ENVIRONMENTAL BIOLOGYAdvice on marking Outcome 3 reportSpecific advice for performance criteria b–fPC b: a description of the method used to extract PCN from a soilsample; a description of a viable and non-viable PCN.PC c: a table with suitable headings showing the total number of cystsper 100 g of at least two soil samples.PC d: a table with suitable headings showing the percentage of viablecysts in at least two soil samples.PC e: a conclusion on how suitable each soil would be for producing acrop of seed potatoes.PC f: evaluation points include:• possible ways of losing PCN cysts during the extractionmethod• the possibility of mistaking a viable PCN for a non-viable one• the reliability of the method used in taking the soil samplefrom a field.Extension workMake exudates from resistant and non-resistant potatoes. Mix thesewith viable cysts and note any differences in number of PCN releasedfrom cysts. A method for making exudate and inducing hatching ofcysts is included in the Technical Guide.As above but vary the exudate, e.g. temperature of mixing, previouslyboiled, vary pH and concentration.Examine a variety of soils for PCN.Test the efficiency of the extraction method by adding a knownnumber of cysts to a soil sample, follow the method given andcalculate the percentage recovered. The extraction method can bevaried and the percentage of cysts recovered monitored.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)4 8ENVIRONMENTAL BIOLOGYReferencesAtkinson H. (1997), ‘The worm in the root!’, Biological SciencesReview, 9(5), 36–38.Council Directive of 8 December 1969 on control of potato cysteelworm (69/465/EEC), Official Journal of the European CommunitiesNumber L323/3 24/12/69.Evans K. A., Harling R. and Dubickas A. (1998), ‘Application of a PCR-based technique to speciate potato cyst nematodes and determine thedistribution of Globodera pallida in ware growing areas’, Aspects ofApplied Biology, 52, 345–350.Evans F. and Haydock P. (1999), ‘Control of plant parasitic nematodes’,Pesticide Outlook, 10(3), 89–128.Marks R. J. and Brodie B. B. (Editors), Potato Cyst Nematodes – Biology,Distribution and Control.AcknowledgementsThe original protocol for this experiment was obtained from the ScottishAgricultural College (SAC), West Mains Road, Edinburgh. Thisinformation and advice from A. Evans and C. Kasperak of SAC aregratefully acknowledged.Information and advice were also obtained from D. Trudgill and A. Holt,Scottish Crop Research Institute (SCRI), Invergowrie.Acknowledgements also to J. Pickup, Scottish Agricultural ScienceAgency (SASA), East Craigs, Edinburgh.This experiment was produced by the SAPS Biotechnology ScotlandProject. Funding for the project was provided by SAPS, Unilever andThe Scottish Office. Support was also provided by Edinburgh University,Quest International, the Scottish CCC, the Higher Still DevelopmentUnit and SSERC.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 4 9ENVIRONMENTAL BIOLOGYTechnical guideMaterials requiredMaterials required by each student/group:large filter paper (185 mm diameter)set of compasses with pencilrulerfilter funnel (top internal diameter about 100 mm)washing bottleglass rodlarge beaker, e.g. 400 cm3binocular microscope (×10 – ×20)compound microscope (×100)piece of acetatelarge conical flask, e.g. 250 cm3pair of fine forcepsmicroscope slidescoverslipsMaterials to be shared:dried soil, gently crushed or rolledbalanceweighing boatssoil sieves with large mesh (550 µm – 850 µm) – mesh no. 30 or 20soil sieves with small mesh (250 µm) – mesh no. 60Preparation of materialsObtaining a suitable soil sample containing viable PCN may present aproblem in some areas. A garden or allotment with a history of growingsusceptible varieties of potatoes (see Teacher/Lecturer Guide) is usuallya good source. In rural areas a local farmer may be willing to providesuitable soil.If taking soil samples from any land you must ensure that all equipmentused and boots worn are clean and could not be contaminated withcysts from a prior sampling site. The distribution of cysts is unlikely tobe uniform. ‘Hot spots’ will occur and so it is important to take severalsamples of about 100 g at intervals throughout the field. Samplingpoints should be chosen randomly and small soil samples lifted using atrowel or the widest cork borer (no. 6 – each bore will give about a 10 gsample).SAPS may be able to supply a limited number of non-viable cysts.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)5 0ENVIRONMENTAL BIOLOGYSoil samples should be dried at room temperature before use. Thisincreases the chances of PCN cysts floating during their extraction fromsoil. If the soil is not fine, it may also need to be passed through ariddle or lumps broken up gently.To make exudate:1. Grow susceptible potato in sand (or sandy soil) for 2–3 weeks.2. Collect and wash roots.3. Cover roots with water and leave for 4–6 hours or overnight in arefrigerator.4. Filter and collect exudate.To induce hatching of cysts:1. Put about 10 cysts in water for 5–7 days.2. Remove all the water and cover with exudate.3. Cysts will start to hatch within 5 days. Remove a few drops ofexudate to a dimpled microscope slide to view nematodes.N.B. New cysts may need to be stored at 4°C for 3–6 months before theywill hatch.Supply of materialsIt is not appropriate to provide all equipment and materials in, forexample, a tray system for each student/group. Equipment andmaterials should be supplied in a way that students have to identify andobtain resources. Normal laboratory apparatus should not be madeavailable in kits but should generally be available in the laboratory. Trayscould be provided containing one type of specialist equipment ormaterials.Disposal of materialsIt is most important that good laboratory practice is carried out duringthis experiment. All materials containing cysts must be autoclaved orsoaked in bleach before being disposed. Suitable precautions are listedin the Student Activity Guide.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5 1ENVIRONMENTAL BIOLOGYPreparing for the activityRead through the Student Activity Guide and consider the followingquestions.Analysis of activityWhat is the aim of the activity?What measurement are you going to take?Are you aware of the size of potato cyst nematode cysts and what theylook like?Are you aware of the differences between viable and non-viable potatocyst nematodes?Are you aware of the precautions you must follow to prevent furtherspread of this parasite?Getting organised for experimental workWhat safety measures are you required to take?In your group decide how the activity will be managed by allocatingtasks to each member. For Outcome 3 it is important that you play anactive part in carrying out the experiment and in collecting results.Recording of dataPrepare a table to record:(i) the total number of cysts in each soil sample(ii) the percentage of viable cysts.You should use a ruler, correct headings and appropriate units whennecessary.EvaluationAre there possible flaws in the extraction process where PCN can be lostfrom the sample, leading to unreliable results?
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)5 2ENVIRONMENTAL BIOLOGYDo you think the procedure involved in taking the soil sample isreliable? Is the sample size (50 g) large enough? (A 500 g sample is usedwhen this procedure is carried out professionally.)Has the filter paper been examined sufficiently or is it possible that cystson it could be overlooked?
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5 3ENVIRONMENTAL BIOLOGYStudent activity guideIntroductionPotato cyst nematodes (PCN), also known as potato cyst eelworms(PCE), are world-wide parasites of potato plants. They originated inSouth America where the Incas practised a seven-course rotation tocontrol them. Being parasites the PCN receive all their nutritionalrequirements from the potato plant, resulting in reduced root and foliargrowth and a reduction in tuber yield. The cost of damage caused byPCN is estimated to be about £43 million each year in the UK alone(1990–1995). This annual cost is increasing as is the incidence of PCN.Like many parasites, PCN have a highly specialised life cycle. The cystsyou are going to isolate are only about 0.5 mm in diameter and maycontain up to 200–600 eggs initially which have larvae coiled up insidethem.0.5 mm0.5 mmevery year a small numberof eggs are releasedspontaneously. Thisnumber increases when asusceptible potato varietyis grown in infected soil.Female becomes attached topotato plant. When fertilisedby male its body swellsand develops into a cyst.Larva emerges from egg, invadesroot and establishes a feeding site. Ifno host plant is available, the larvadies within days.Cyst - light brown in colour.Contains 200-600 eggs.Can remain dormant in soilfor up to 30 years.Egg containingcoiled up larva.Cyst – light brown in colour.Contains 200–600 eggs. Canremain dormant in soil for upto 30 years.Every year a small numberof eggs are releasedspontaneously. Thisnumber increases when asusceptible potato varietyis grown in infected soil.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)5 4ENVIRONMENTAL BIOLOGYInfection of potato plants by PCN has several effects:(i) Even moderately low population densities (about 5 eggs per gramof soil) will reduce yields and high populations (200–2000 eggs pergram of soil) may result in complete crop loss.(ii) As a result of infection, plants have a stunted root system makingthem more susceptible to drought.(iii) Secondary invaders, e.g. fungi, can enter the root system morereadily.The main means of passive transmission of PCN are through the plantingof infected potatoes, i.e. potatoes grown on infected land, and by themovement of contaminated soil, e.g. that adhering to farm machinery.They are mainly controlled by using a combination of the following:(i) Crop rotation (long rotations allow natural population decline)(ii) Use of resistant varieties which inhibit PCN multiplication(iii) A type of pesticide known as nematicides (affect the nervoussystem of juveniles which prevents juveniles locating a host plant).Equipment and materialsMaterials required by each student/group:large filter paper (185 mm diameter)set of compasses with pencilrulerfilter funnel (top internal diameter about 100 mm)washing bottleglass rodlarge beaker, e.g. 400 cm3binocular microscope (×10 – ×20)compound microscope (×100)piece of acetatelarge conical flask, e.g. 250 cm3pair of fine forcepsmicroscope slidescoverslipsMaterials to be shared:dried soil, gently crushed or rolledbalanceweighing boatssoil sieves with large mesh (550 µm – 850 µm) – mesh no. 30 or 20
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5 5ENVIRONMENTAL BIOLOGYsoil sieves with small mesh (250 µm) – mesh no. 60detergent with dropperPrecautions required to be takenAs potato cyst nematodes are a serious pest to an economicallyimportant food crop, the precautions listed below must be followed.1. If taking soil samples from any land you must ensure that allequipment used and boots worn are clean and could not becontaminated with cysts from a prior sampling site.2. Find out if sludge from your local sewage treatment plant is spreadon agricultural soil. If so, all possible precautions should befollowed to prevent viable cysts from being washed down the sink.3. After use, all apparatus such as sieves and glassware should beautoclaved or soaked in bleach overnight before being washed.Such treatment will kill viable cysts.4. Wipe up spillages with a paper towel and place in a bin.5. Care must be taken to avoid cross-contamination of samples.InstructionsN.B. For successful extractions, cysts must be clean and previouslydried in the soil at room temperature.1. Weigh out 50 g of the dried soil. The soil sample has a history ofbeing used for growing potatoes. Break up any small lumps gentlywith the end of a glass rod.2. Collect the two soil sieves, fitting the one with the larger mesh sizeon top. Place the sieves above a bucket or polythene bag and addthe soil sample to the top sieve.3. Sift the dry soil for 3–4 minutes.4. Wash the sieves under a fast running tap. Cysts will not passthrough the finer sieve so it can be washed on its own under thetap. When washing the larger mesh sieve always place the finermesh sieve beneath it.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)5 6ENVIRONMENTAL BIOLOGYIn the instructions that follow, treat the contents of each sieveseparately. Each group of students should therefore form twosmaller groups, one working with the soil in the large mesh sieve,the other with the soil in the small mesh sieve.5. Away from the sink, wash outthe contents of your allocatedsieve into a beaker with the helpof a wash bottle.To do this, hold the sieve almostat right angles above the beakerand with a wash bottle project astream of water on to what wasthe lower side of the sieve.Slowly rotate the sieve whiledoing this. Then, turning it theright way up, wash finalcontents from the sieve. Donot now wash sieves in the sink– see precautions.6. Allow the soil/water mixture to settle until little movement ofmaterial is occurring (10 minutes).7. Meanwhile, using a pair ofcompasses and a pencil, draw fourconcentric circles on a large filterpaper (as shown in the diagram).Ensure the circles drawn arecomplete and prominent. Draw astraight line from the centre to theedge of the filter paper.8. Fold this filter paper twice and fit itinto a filter funnel. Sit the funnelon top of a large conical flask.9. Once the contents of the beaker have settled, decant quickly intothe filter paper without disturbing the sunken soil. Whiledecanting, rotate the beaker slowly so that any floating debris stuckto the sides gets washed into the filter paper.washbottlesievesoil sample185 mm15 mm
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5 7ENVIRONMENTAL BIOLOGY10. Add a drop of detergent to the soil/water mixture while it isfiltering. This encourages any cysts present to migrate to the sidesand stick to the paper.11. Using a high pressure flow of water, add about 200 cm3to thebeaker containing the soil. Allow to settle and decant as beforeinto the filter paper.12. Once filtration is complete remove the filter paper from thefunnel, unfold it and place overnight in a humid, airtight container.This ensures that the cysts will burst easily.13. On the next day, place the filter paper on a suitable surface (e.g. apiece of acetate) and examine under the binocular microscope.Starting at the straight line in the outermost circle, examine thiscircle for cysts. Repeat this procedure for the other circles on thefilter paper.Potato cyst nematode cysts are only 0.5 mm in diameteron average. However, they are easily detected by theirshape and colour – perfectly spherical apart from asmall ‘neck’ (rather like a gourd or a sphericaldecoration commonly put on a Christmas tree). Theyvary from being orange and copper coloured to a dull dark brown.Warning: Other cysts may be present, e.g. cereal cyst nematode(these are lemon shaped).14. With a pair of fine forceps remove any cysts from the filter paperand place in a droplet of water on a microscope slide. Theconcentric circles drawn previously should help to ensure theentire filter paper is scanned although most cysts should be foundin the outermost circle. Count the total number of cysts found onthe filter paper. Add this to the number found on the filter paperfrom the other sieve of the same soil sample.15. Select at random several cysts and place them far enough apart ona few microscope slides so that each can be covered by a separatecoverslip. Add a drop of water to each cyst and cover each onewith a coverslip.16. Examine each cyst in turn under a microscope (×100 totalmagnification). Whilst viewing a cyst press down gently on thecoverslip. This will cause the cyst to burst and release its contents.Look in particular at any larvae whose egg case has burst. If the
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)5 8ENVIRONMENTAL BIOLOGYegg case does not burst you will see capsule-shaped objects as inthe diagram of the life cycle. Determine the number of cystscontaining viable larvae.N.B. Do not attempt to burst open all the eggs. A cyst just needs tocontain ONE viable larvae for it to be scored as viable. If cysts arecompletely empty, assume they are non-viable.17. Calculate:(i) the total number of cysts per 100 g of your soil sample (youstarted with a 50 g sample).(ii) the percentage of viable cysts in your random sample of cysts.18. Compare the soil sample you have just examined with one with adifferent history for growing potatoes.19. Present your results in a table with suitable headings. Draw a barchart with the axes labelled appropriately to show the resultsgraphically.The experiment you have just done is a simplified, scaled-down versionof a test carried out routinely on fields intended for the production ofseed potatoes. If even one viable potato cyst nematode is found in a500 g sample then the field cannot be used to provide seed potatoes.N.B. 1. This experiment is done for educational purposes only andshould not be used as a basis for any agronomic decisionsdue to the relative inexperience of the testers.2. Soil and any equipment used in the experiment must now beautoclaved to kill any PCN cysts. Do not dispose of any soilsamples by returning them to land from which they did notoriginate.Viable larvae will uncoil completelywhen the egg case bursts. Their‘skin’ will be smooth and free ofany sudden indentations. Non-viable larvae will have foldsand ‘kinks’ in their ‘skin’.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 5 9BIOTECHNOLOGYACTIVITY 9Unit: Biotechnology (AH): Use of Micro-organisms: Stages ofgrowthTitle: Growth curve: Determination of doubling time and growthrate constantTeacher/lecturer guideType and purpose of activityThis experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the stages of growth ofmicrobes in culture, turbidometric measurement of cell growth andgrowth rate constants• develop problem solving skills and in particular Outcome 2performance criteria:(a) relevant information is selected and presented in theappropriate format(b) information is accurately processed using calculations whereappropriate.Background informationIn industry, it is important to be able to determine the growth rate of agiven micro-organism and understand the factors that affect it in orderto generate maximum product by the most economic means. Theproduct may be a metabolite produced at a given stage of the growthcycle or it may be the organism itself, e.g. the production of yeastbiomass to be used as starter cultures for brewing or baking, or as thestarting point for autolysis which produces a huge variety of foodflavourings.As the number of cells in a microbial culture increases, the turbidity(cloudiness) of the culture increases. Turbidity is caused by thesuspended cells scattering light and it may be measured using acolorimeter. Absorbance increases as the cell concentration increases
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)6 0BIOTECHNOLOGYgiving a convenient, rapid and accurate method of measuring cellgrowth rates.This method cannot, however, distinguish between live and dead cells.A growth curve generated by this method over the time suggested willnot demonstrate the death or senescent phase. Viable counts wouldhave to be carried out.Absorbance is plotted on a graph against time. Doubling of absorbanceindicates doubling of the number of cells and the time taken for this tooccur can be read from the graph.In this experiment students will create a growth curve of absorbanceversus time, then use it to calculate doubling time and growth rateconstant using absorbance as the measure of growth.Classroom managementObtaining results for a growth curve cannot be managed in one lesson.This practical has been designed with the aim of ease of collection ofdata and production of a classic growth curve shape showing lag phase,exponential phase, stationary phase and eventually death phase if theculture is left long enough or viable counts are measured.Medium is inoculated with a very small quantity of yeast late in theafternoon then samples are taken three times per school day for thenext three or four days (early morning, lunchtime and late afternoon).Timing is not critical but time of sampling should be recorded so thathours of incubation can be calculated.Samples do not have to be read immediately – they can be placed insterile Bijoux bottles, tubes or universals, refrigerated and theabsorbance read when the time is convenient, although preferablywithin 24 hours. The yeast cells will settle so it is very important toshake gently to suspend the cells before reading the absorbance.Students working as part of a group could arrange a rota for removal ofsamples.A number of factors are important.• Very small inoculum – to allow good demonstration of lag phase.• Timing – lag phase has been best observed by inoculating themedium late in the afternoon.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 6 1BIOTECHNOLOGY• Yeast type – do not use fast acting yeasts as they show a very short lagphase, if any.• Cultures must be gently agitated before removing a sample to ensurethat the cells are in suspension.• Samples must be well suspended before taking a reading.Supply of materialsIn order to satisfy the core skill in problem solving, students will berequired to identify and obtain resources required for themselves.Further advice on supply of material is given in the Technical Guide.Advice on marking Outcome 3 reportSpecific advice for performance criteria b–fPC b: to include description of how the low inoculum concentration isachieved; method of sampling; method of measuringabsorbance.PC c: a table of results with appropriate headings and units showingthe time and date of sampling, hours of growth and absorbance.PC d: a graph of absorbance on the y-axis and hours on the x-axis. Lagphase, log phase and time of stationary phase should belabelled. Indication of measurement of generation (doubling)time should be made on the graph (i.e. the time taken for theabsorbance to double).PC e: growth rate constant is calculated using the generation timedetermined from the graph.PC f: evaluation points include:• accuracy of inoculum concentration• mixing of culture before removal of samples• suspension of cells before reading absorbance• control of temperature• determination of doubling time from graph• usefulness of semi-logarithmic graph paper in plotting and/oranalysing results.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)6 2BIOTECHNOLOGYExtension workThe effects on the growth curve and the growth rate constant of varyingthe growth media.The effects on the growth curve and growth rate constant of varying theincubation temperature.The effects of different concentrations of starter culture on the length ofthe lag and log phases of growth and initiation time of stationary phase.The effects on the growth curve of keeping the inoculum underdifferent conditions before inoculation, e.g. in the fridge.Comparison of growth curves and growth rate constants for differentmicro-organisms or types of dried yeast in the same media.Comparison of different methods of enumerating micro-organisms (e.g.haemocytometer and viable count) to generate a growth curve.ReferencesIain S. Hunter (2000), Biology: Biotechnology Student Monograph(Advanced Higher), Learning and Teaching ScotlandAcknowledgmentThis experiment was produced by the SAPS Biotechnology ScotlandProject. Funding for the project was provided by SAPS, Unilever andThe Scottish Office. Support was also provided by Edinburgh University,Quest International, Learning and Teaching Scotland, the Higher StillDevelopment Unit and SSERC.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 6 3BIOTECHNOLOGYTechnical guideMaterials requiredMaterials required by each student/group:5 cm3sterile yeast glucose broth as blank99 cm3sterile yeast glucose broth in flaskdried yeast (not fast acting)weighing boatspatula10 cm3sterile water (if balance is accurate to 0.01 g)100 cm3sterile watersterile 1 cm3pipettediscard jar containing 2% stericolsemi-log graph paperMaterials to be shared:waterbath or incubator at 30°Cbalance (accurate to 0.001 g preferably, or 0.01 g)colorimeter (440 nm)Preparation of materialsYeast glucose broth (for 1 litre medium)20 g glucose20 g bactopeptone10 g yeast extract0.1 M sulphuric acid or 0.5 M sodium hydroxidedistilled waterInstructions1. Wear a lab coat.2. Weigh glucose, bactopeptone and yeast extract into a beaker.3. Add distilled water to 1 litre mark.4. Stir thoroughly and adjust to pH 6.5. Dispense volumes into required containers for autoclaving.6. Autoclave for time and temperature appropriate to medium.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)6 4BIOTECHNOLOGYNotes• Medium for blanks can be kept in sterile Bijoux bottles or small steriletest tubes (plugged or covered) and can be refrigerated and usedover the four days taken to generate the growth curve.• Media should be made up, dispensed into Bijoux bottles, test tubes orflasks (covered or plugged) then sterilised immediately byautoclaving.• Tins of traditional dried yeast are better as sachets of yeast tend to beof the fast-acting variety and do not demonstrate lag phase so well.• When samples have been read, the yeast suspension should bedisposed of into a discard jar containing 2% stericol and the cuvettewashed with detergent and hot water.• Digital colorimeters, e.g. WPA CO75 or Harris S-Range colorimeter,are best used for this experiment. Older colorimeters may not besensitive enough.Supply of materialsIt is not appropriate to provide all equipment and materials in, forexample, a tray system for each student/group. Equipment andmaterials should be supplied in a way that students have to identify andobtain resources. Normal laboratory apparatus should not be madeavailable in kits but should generally be available in the laboratory. Trayscould be provided containing one type of specialist equipment ormaterials.
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 6 5BIOTECHNOLOGYPreparing for the activityRead through the Student Activity Guide and consider the followingquestions.Analysis of activityWhat is the aim of the activity?What measurements are you going to make?How will you record these measurements?How will you determine the information you require to make the finalcalculation?What constant will you calculate?Getting organised for experimental workIn your group decide how the activity will be managed by allocatingtasks to each member. It is very important that samples are removed atleast three times per day.Recording of dataPrepare tables and semi-logarithmic graph paper to record your groupresults.You should use a ruler, correct headings and appropriate units.EvaluationHow effective were the methods which you used?What were the limitations of the equipment?What were the sources of error?What possible improvements could be made to the experiment?What is the benefit of plotting results on semi-logarithmic graph paper?What is the economic importance of the process which you are studyingand the calculations which you will make?
FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY)6 6BIOTECHNOLOGYStudent activity guideIntroductionGrowth is the process during which living organisms synthesise newchemical components for the cell and as a result they usually increase insize. In unicellular organisms, such as bacteria and yeast, growth leadsto cell division and consequently an increase in population size. Thegrowth of a population of single-celled micro-organisms grown in aclosed environment typically shows four stages: lag phase; exponentialphase; stationary phase; death phase.The lengths and characteristics of these phases will depend uponfactors such as the nature of the growth medium and temperature ofincubation.In industry, it is important to understand the factors which affect thegrowth rate of a given micro-organism in order to generate maximumproduct by the most economic means. For example, if the desiredproduct is a secondary metabolite such as an antibiotic which isproduced when the organism has stopped growing, the manufacturerwill want to provide optimum conditions for the culture to reachmaximum numbers in stationary phase in the shortest time possible.In some cases, the product is the organism itself, e.g. the production ofyeast biomass to be used as starter cultures for brewing or baking, or asthe starting point for autolysis which produces a huge variety of foodflavourings.Growth of a population can be measured using the following methods:Cell counts: total numbers of cells are counted directly using amicroscope and a special slide called a haemocytometer.Dilution plating: the culture is serially diluted and a known volume ofeach dilution plated out and incubated. Resulting colonies are countedgiving a measure of viable numbers of cells in the original population.Turbidometric methods: Cell density is measured using a colorimeter.This is a photometric method which measures the light scattered by thecells in suspension. Increase in cell density is an extremely accuratemethod of measuring cell growth rates.