biology tcm4 123674 (1)

FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1

INTRODUCTION

Revised practicals for mandatory units

This 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 unit

A 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 PR ACTICAL ACTIVITIES (AH BIOLOGY)2

INTRODUCTION

Experimental work

One 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 an

appropriate format.d. Recorded experimental information is analysed and presented in

an appropriate format.e. Conclusions drawn are valid.f. The experimental procedures are evaluated with supporting

argument.

Purpose

A 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.

Structure

Teacher/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


INTRODUCTION

student 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


INTRODUCTION

activity’ 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 3

Arrangements 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 writing

Students 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


INTRODUCTION

advice 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 reports

The ‘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.


CELL AND MOLECULAR BIOLOGY

ACTIVITY 1

Unit: Cell and Molecular Biology (AH): Structure, function andgrowth of prokaryotic and eukaryotic cells

Title: Staining a root tip and calculating its mitotic index

Teacher/lecturer guide

Type and purpose of activity

This 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 2

performance criteria:

(b) information is accurately processed, using calculations whereappropriate

(d) experimental procedures are planned, designed and evaluatedappropriately.

Background information

In 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.



Preparation 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 management

Students 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 report

Specific advice for performance criteria b–fPC b: a description of the preparation of the root tip(s) and the

method(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).



PC 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 work

Try 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.

Acknowledgements

Information 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 PR ACTICAL ACTIVITIES (AH BIOLOGY)1 0


Technical guide

The class will be varying either plant material or stain for this activity.The list of materials required will vary depending on this decision.

Materials required

Materials 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 material

is being investigated)small beaker of water and droppermicroscope slidescoverslipsfine forcepsdissecting needlescissorssoft tissue paperrulerfine threaddropping bottle of lactopropionic orcein and/or (see below) dropping

bottle of toluidine bluegarlic clove with suitable roots and/or (see below) hyacinth bulb with

suitable roots

Materials to be shared:

waterbath at 60°Cmarker pentimerdropping bottle of 50% glyceroldropping bottle of 70% ethanollens tissue

Preparation of materials

If 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 1


Hyacinth 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 cm3 0.1 M citric acid + 10 cm3 0.2 Mdisodium hydrogen phosphate + 8 cm3 distilled water).

Fixing the rootsThis stage is required only if suitable roots are available but they are tobe stained at a later date.

Mix 6 cm3 absolute alcohol with 2 cm3 glacial 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 materials

It 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.



Preparing for the activity

Read through the Student Activity Guide and consider the followingquestions.

Analysis of activity

What 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 data

Prepare a table to record your results. You should use a ruler andappropriate headings.

Evaluation

If 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.)



Student activity guide

Introduction

You 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 materials


gloves and eye protectioncompound microscope (×100 – ×400 magnification)small beaker of 1 M hydrochloric acid (2 will be required if plant material

is being investigated)small beaker of water and droppermicroscope slidescoverslipsfine forcepsdissecting needlescissorssoft tissue paperrulerfine threaddropping bottle of lactopropionic orcein and/or (see below) dropping

bottle of toluidine bluegarlic clove with suitable roots and/or (see below) hyacinth bulb with

suitable roots


waterbath at 60°Cmarker pentimerdropping bottle of 50% glyceroldropping bottle of 70% ethanollens tissue

Wear 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.



Instructions

Either 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 cm3 of 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.



9. 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.



ACTIVITY 4

Unit: Cell and Molecular Biology (AH): Applications of DNAtechnology

Title: Gel electrophoresis of DNA treated with restrictionenzymes




• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of cutting DNA with

restriction enzymes• develop problem solving skills and in particular Outcome 2


(c) conclusions drawn are valid and explanations given aresupported by evidence



This 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.



In 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.


Following 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 run

battery (4 × 9 V) 36 V about 2 hours

*regulated power pack 30 V about 2.5 hours

unregulated 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 materials

In 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.




Specific 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.)


• 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.



• 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?

References

Investigating 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.uk

Acknowledgement

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.



Technical guide

Materials required


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 pen

Day 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 tissue

Day 3 – tank containing your gel from previous lessonstain (10 cm3)gloveseye protection


Day 1 – waterbath at 37°CDay 2 – bottle of TBE buffer


Preparation of materials supplied by the kit

Rehydrating 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.



Two 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 cm3 will be required per group (11–12 cm3 todissolve 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 cm3 of 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 kit

Making 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 materials




Disposal of materials

All 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.








Are you familiar with how the restriction enzymes act on DNA?

Are you aware of what is happening during electrophoresis?

Getting organised for experimental work


Are you familiar with the microsyringe and how to deliver a set volumeusing it?

Recording of data

Prepare 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.

Evaluation

Why 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?




Introduction

This 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 stained

If 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 enzyme

called HindIII– by mixing a third sample of DNA with both of these enzymes.

DNA cut withrestrictionenzymes

electriccurrent



Each 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 EcoRI

The 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.



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 pen

Day 2 – electrical supply2 electric wires with crocodile clipsenzyme tubes in the float from the previous lesson

DNAdoublehelix

DNA cutintofragments

G

C

T

A G C T T A A

C G A A T T

G

C G A C

C T G

G

C

T

A G C T T A A

C G A A T T

G

C G A C

C T G



vial of loading dyegel in a plastic tank with comb, covered in buffer solutionmicrosyringe and 4 tipspiece of black card2 pieces of carbon fibre tissue

Day 3 - tank containing your gel from previous lessonstain (10 cm3)gloveseye protection


Day 1 – waterbath at 37°CDay 2 – bottle of TBE buffer

Instructions

Preliminary exercise

This 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



Microsyringe 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 DNA

1. 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



the 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 fragments

1. If not already done, cover the gel with about 20 cm3 of 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 DNA

buffer solution

gel

microsyringe tip



6. 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 fibre

wells

buffer solution



Day 3 – Staining the DNA

1. Return the buffer solution covering the gel to its originalcontainer.

2. Pour about 10 cm3 of 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.

Lanes 1 2 3 4

DNAbands

largest

smallest

loading dye

wells



Lane 1 Lane 2 Lane 3 Lane 4Contents Undigested DNA digested DNA digested DNA digested

DNA by restriction by restriction by bothenzyme, enzyme, restrictionEcoRI HindIII enzymes

No. 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 3.548

3,530 3.548 2,027 3.307 2,027 3.307564 2.751 1,904 3.280125 2.097 1,584 3.200

1,375 3.138947 2.976831 2.920564 2.751125 2.097

6. 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).



Unit: Cell and Molecular Biology (AH): Molecular interactions incell events: Catalysis

Title: The effect of competitive and non-competitive inhibitorson the enzyme β-galactosidase




• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the effect of competitive

and non-competitive inhibitors on enzyme activity• develop problem solving skills and in particular Outcome 2


(c) conclusions drawn are valid and explanations given aresupported by evidence



The enzyme β-galactosidase catalyses the following reaction:

LACTOSE GLUCOSE + GALACTOSE

The chemical ONPG (o-nitrophenyl β-D-galactopyranoside) is alsodegraded by the enzyme:

ONPG ONP + GALACTOSE

The 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 active

ACTIVITY 8

β-galactosidase

β-galactosidase



site. 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.


Students 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.


Specific advice for peformance criteria b–fPC b: to include a description of the contents of the various cuvettes

set 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.



PC 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.


• 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 work

Substitute 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/V0 is 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).



References

Adds, 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.



Technical guide

Materials required

Materials required by each student/group:6 cuvettes (or test tubes if suitable colorimeter is used)2 boiling tubesbeaker of crushed ice6 × 1 cm3 droppers10 cm3 syringe6 cm3 ONPG stock solution (3 × 10-2 M in buffer)40 cm3 buffer (0.1 M potassium phosphate, pH 8)15 cm3 20% galactose in buffer5 cm3 I

2/KI solution in buffer

25 cm3 distilled watereye protectiongloves

Materials to be shared:colorimeter (420–440 nm filter)1 cm3 dropperdistilled waterβ-galactosidase stock solution


The buffer: 0.1 M K2HPO4 adjusted 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 × 10-2 M in buffer. Each student/group willrequire 6 cm3. For every 10 cm3 required, weigh out 0.09 g and dissolvein 10 cm3 buffer. 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 cm3 buffer.It dissolves readily.



I2/KI solution: Each student/group will require about 5 cm3. Dissolve

0.3 g iodine and 1.5 g potassium iodide in 100 cm3 water to make a stocksolution (this will keep for months stored in a dark glass bottle). Take1 cm3 of this stock solution and make up to 80 cm3 with 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.







What is being varied in the activity?

What variables must be kept constant?


Why should the enzyme activity be measured without either inhibitorboth at the beginning and at the end of the experiment?



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 data

Prepare tables to record your group results.

You should use a ruler, correct headings and appropriate units.

Evaluation

Has 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?




Introduction

Inhibitors 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




Materials required by each student/group:6 cuvettes (or test tubes if suitable colorimeter is used)2 boiling tubesbeaker of crushed ice6 × 1 cm3 droppers10 cm3 syringe6 cm3 ONPG stock solution (3 × 10-2 M in buffer)40 cm3 buffer (0.1 M potassium phosphate, pH 8)15 cm3 20% galactose in buffer5 cm3 I2/KI solution in buffer25 cm3 distilled watereye protectiongloves

Materials to be shared:colorimeter (420–440 nm filter)1 cm3 dropperdistilled waterβ-galactosidase stock solution

Instructions

Wear eye protection and gloves throughout this experiment to avoiddirect skin and eye contact with some of the chemicals used.

1. Put 20 cm3 of 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 cm3 of the stock ONPG solution with 9.5 cm3 of 0.1M buffer(pH 8). Label ×20 dilution.

3. Put 2 cm3 of buffer and 1 cm3 of this ×20 diluted ONPG solutioninto a cuvette. Mix by inverting the cuvette 2–3 times. Zero thecolorimeter with this solution.



4. Add 0.5 cm3 of 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) in

the 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 cm3

7. 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 cm3 of 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.



You are now going to investigate:(i) the effect of iodine solution (another inhibitor) on the activity of

the enzyme(ii) the effect of increasing the ONPG concentration in the presence of

the 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 cm3 of 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.


ENVIRONMENTAL BIOLOGY

ACTIVITY 5

Unit: Environmental Biology (AH): Symbiotic relationships(Parasitism)

Title: Isolating and examining cysts of potato cyst nematodes




• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of parasitism and more

specifically 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



An 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 can

be collected and examined at any time of year(iii) controlling PCN is expensive, complicated and an ever increasing

problem.

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



resistant 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.


Obtaining 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 materials

In 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.




Specific advice for performance criteria b–f

PC 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 extraction

method• the possibility of mistaking a viable PCN for a non-viable one• the reliability of the method used in taking the soil sample

from a field.

Extension work

Make 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.



References

Atkinson 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.

Acknowledgements

The 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.



Technical guide

Materials required


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 cm3

binocular microscope (×10 – ×20)compound microscope (×100)piece of acetatelarge conical flask, e.g. 250 cm3

pair of fine forcepsmicroscope slidescoverslips


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. 60


Obtaining 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.



Soil 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 a

refrigerator.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 of

exudate 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 materials



It 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.







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?



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 data

Prepare 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.

Evaluation

Are there possible flaws in the extraction process where PCN can be lostfrom the sample, leading to unreliable results?



Do 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?




Introduction

Potato 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 mm

0.5 mm

every 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.



Infection 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 nervous

system of juveniles which prevents juveniles locating a host plant).



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 cm3

binocular microscope (×10 – ×20)compound microscope (×100)piece of acetatelarge conical flask, e.g. 250 cm3

pair of fine forcepsmicroscope slidescoverslips


dried soil, gently crushed or rolledbalanceweighing boatssoil sieves with large mesh (550 µm – 850 µm) – mesh no. 30 or 20



soil sieves with small mesh (250 µm) – mesh no. 60detergent with dropper

Precautions required to be taken

As 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.

Instructions

N.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.



In 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.

washbottle

sieve

soil sample

185 mm

15 mm



10. 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 cm3 to 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



egg 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 folds

and ‘kinks’ in their ‘skin’.


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ACTIVITY 9

Unit: Biotechnology (AH): Use of Micro-organisms: Stages ofgrowth

Title: Growth curve: Determination of doubling time and growthrate constant




• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the stages of growth of

microbes in culture, turbidometric measurement of cell growth andgrowth rate constants


(a) relevant information is selected and presented in theappropriate format

(b) information is accurately processed using calculations whereappropriate.


In 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


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giving 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.


Obtaining 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 the

medium late in the afternoon.


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• 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 materials



Specific advice for performance criteria b–fPC b: to include description of how the low inoculum concentration is

achieved; 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.


• 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/or

analysing results.


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Extension work

The 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.

References

Iain S. Hunter (2000), Biology: Biotechnology Student Monograph(Advanced Higher), Learning and Teaching Scotland

Acknowledgment

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, Learning and Teaching Scotland, the Higher StillDevelopment Unit and SSERC.


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Technical guide

Materials required


5 cm3 sterile yeast glucose broth as blank99 cm3 sterile yeast glucose broth in flaskdried yeast (not fast acting)weighing boatspatula10 cm3 sterile water (if balance is accurate to 0.01 g)100 cm3 sterile watersterile 1 cm3 pipettediscard jar containing 2% stericolsemi-log graph paper


waterbath or incubator at 30°Cbalance (accurate to 0.001 g preferably, or 0.01 g)colorimeter (440 nm)


Yeast glucose broth (for 1 litre medium)

20 g glucose20 g bactopeptone10 g yeast extract0.1 M sulphuric acid or 0.5 M sodium hydroxidedistilled water

Instructions

1. 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.


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Notes

• 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 materials



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How will you record these measurements?

How will you determine the information you require to make the finalcalculation?

What constant will you calculate?


In 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 data

Prepare tables and semi-logarithmic graph paper to record your groupresults.


Evaluation

How 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?


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Introduction

Growth 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.


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In this practical, you will produce a growth curve of absorbance againsttime for a culture inoculated with a known dry mass of Saccharomycescerevisiae (bakers’ yeast) then grown over several days. From this youwill be able to calculate generation time and a growth rate constant.



5 cm3 sterile yeast glucose broth as blank99 cm3 sterile yeast glucose broth in flaskdried yeast (not fast acting)weighing boatspatula10 cm3 sterile water (if balance is accurate to 0.01 g)100 cm3 sterile watersterile 1 cm3 pipettediscard jar containing 2% stericolsemi-log graph paper


water bath or incubator at 30°Cbalance (accurate to 0.001 g preferably, or 0.01 g)colorimeter (440 nm)

Instructions

1. Start this experiment late afternoon at the start of a week.

2. Using aseptic technique, add 0.025 g dried yeast to 100 cm3 steriledistilled water at 30°C. Shake gently to ensure that the cells areevenly distributed and suspended.

(Note: if your balance is not sensitive enough to measure out sucha small quantity, add 0.25 g yeast to 10 cm3 sterile distilled water,mix well then aseptically withdraw 1 cm3 and add to 99 cm3 steriledistilled water.)

3. Using aseptic technique, dilute 100 times by adding 1 cm3 to99 cm3 sterile broth in a flask. This should give a startingconcentration of 0.0025 g/l for your growth curve.

4. Using sterile medium as the reference, calibrate the colorimeter(i.e. set it to zero).


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Note: keep this reference medium (the blank) in the refrigeratorthroughout the experiment.

5. Shake the flask containing the yeast culture gently to distribute thecells evenly. Using aseptic technique, withdraw a 5 cm3 sample.

6. Measure the absorbance of the sample you have just withdrawn.Record date, time and absorbance in the table.

7. Incubate at 30°C.

8. Repeat Instructions 5–7 three times per day for the next three days(early morning, lunchtime and late afternoon if possible). If it isnot possible to measure the absorbance at the time of taking thesample, place it in a sterile container, label with initials, date andtime and refrigerate until convenient to do so, preferably within 24hours. Make sure that the yeast is fully suspended before readingthe absorbance.

9. Present your results as a graph with suitable scales and axes usingsemi-logarithmic graph paper.

10. Identify on your graph the lag, log and exponential phases of thegrowth curve.

11. From the exponential (log) phase of growth curve, work out thetime in hours taken for the absorbance and hence the populationsize to double.

12. Calculate the growth rate constant.


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Supplementary student information

Calculation of growth rate constant

Growth rate constant, k, is a measure of the number of generations (thenumber of doublings) that occur per unit of time in an exponentiallygrowing culture.

ln 2 = k

g

where ln 2 is the natural log of 2 (determine this from your calculator)and g is the time in hours taken for the population to double during theexponential phase of growth.


BIOTECHNOLOGY

ACTIVITY 10

Unit: Biotechnology (AH): Use of Micro-organisms: Stages ofgrowth

Title: Growth Curve 2: Determination of yeast concentrationfrom a standard curve




• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the use of turbidometric

methods in the preparation of a standard curve• develop knowledge and understanding of the use of such a standard

curve in measuring the changes in cell concentration of microbesgrowing in a liquid culture


(a) relevant information is selected and presented in theappropriate format

(b) information is accurately processed using calculations whereappropriate



This experiment is a continuation of the experiment Growth curve:determination of doubling time and growth rate constant. Students willcreate a standard curve of absorbance against yeast concentration (drymass grams per litre) from known concentrations of dried yeast insterile broth. Commercially available dried yeast is used to produce theyeast suspensions. From the graph they will determine theconcentration, in grams per litre, of yeast produced over time usingtheir absorbances obtained in the previous experiment.


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A number of factors are important:

• the medium should be the same as that used for growing the yeast inthe earlier experiment

• the medium should be sterilised in the same way to ensure it iscomparable

• bottles must be gently agitated to ensure the cells are well suspendedbefore carrying out serial dilutions

• students must be advised to make their dilutions accurately• ensure the cells are well suspended before taking a sample for

reading• the medium should be kept cool throughout the experiment to

prevent yeast growth• ideally it would be best to weigh out the dried yeast and suspend it in

an appropriate volume of medium. Most school balances, however,are not sensitive enough for this. The experiment has therefore beendesigned to make up a standard dilution from which further dilutionsare made to give the required concentrations. Careful attentionshould be paid to making the dilutions in order to achieve a goodstandard curve.

It can take up to 15 minutes for the cells in the standard dilution in step2 to resuspend fully. During this time students could carry out steps 3and 4.

Supply of materials



Specific advice for performance criteria b–fPC b: to include description of how the different concentrations are

achieved and method of measuring absorbance

PC c: tables of results with appropriate headings and units showingthe concentration of yeast in g/l and absorbance at 440 nm


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PC d: a graph of absorbance at appropriate wavelength on the y-axisagainst concentration in g/l on the x-axis to create a standardcurve

PC e: concentration in g/l of yeast is determined using the standardcurve


• accuracy of yeast concentrations• accuracy in measuring out volumes of medium and serial

dilutions• mixing of culture before removal of samples• suspension of cells before reading absorbance• control of temperature• accuracy in generating the standard curve• accuracy in reading from the standard curve.

Extension work

The effects on changes in cell concentration of varying the growthmedia.

The effects on changes in cell concentration of varying the incubationtemperature.

The effects of different concentrations of starter culture on changes incell concentration with time.

The effects on changes in cell concentration of keeping the inoculumunder different conditions before inoculation, e.g. in the fridge, roomtemperature.

Comparison of changes in cell concentration for different types of driedyeast in the same media.

Comparison of changes in cell concentration with changes in total/viablecount in a growing culture.


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References

Iain S. Hunter (2000), Biology: Biotechnology Student Monograph(Advanced Higher), Learning and Teaching Scotland.

Acknowledgement



BIOTECHNOLOGY

Technical guide



100 cm3 sterile yeast glucose broth in flask (or bottle)11 large test tubes (or universals)test-tube rackdried yeast (not fast acting)weighing boatspatula4 × 10 cm3 syringes (or pipettors with tips)1 cm3 pipettecuvettesdiscard jar containing 2% stericolgraph papermarker pen


balance (accurate to 0.01 g)colorimeter (440 nm) – see Notes belowcrushed ice (optional)


Yeast glucose broth (for 1 litre medium)

20 g glucose20 g bactopeptone10 g yeast extract0.1 M sulphuric acid or 0.5 M sodium hydroxidedistilled water

Instructions

1. 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 100 cm3 volumes into conical flasks or bottles forautoclaving.

6. Autoclave for time and temperature appropriate to medium.


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Notes

• Media should be made up, dispensed into flasks or bottles (coveredor plugged) then sterilised immediately by autoclaving.

• Tins of traditional dried yeast should be used – not fast-acting types.• Digital colorimeters, e.g. WPA CO75 or Harris S-Range colorimeter,

are best used for this experiment. Older colorimeters may not besensitive enough.

• If the experiment is to be carried out in a warm room, crushed icemay be required to keep the tubes cool while carrying out theexperiment. Providing sterile media which has been stored in afridge will also help. These measures will reduce chances of celldivision.

Supply of materials



Syringes, pipettes and cuvettes can be washed with detergent and hotwater for reuse. Tubes containing broth should be autoclavedappropriately or soaked in 2% stericol for 24 hours to sterilise.


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How will you record these measurements?

How will you determine concentration of yeast cells in a growingculture?


In your group decide how the activity will be managed by allocatingtasks to each member.

Recording of data

Prepare tables and graph paper to record your group results.


Evaluation

How effective were the methods which you used?

What is the significance of using g/l as the measurement ofconcentration?




What is the economic importance of the process which you are studyingand the calculations which you will make?


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Introduction

Stages of growthIn industry it is often important to determine the actual concentrationof cells in a growing culture. This may involve counting total numbersof cells, numbers of viable cells or cell concentration in terms of drymass.

In the previous experiment, Growth curve: determination of doublingtime and growth rate constant, you created a growth curve ofabsorbance at 440 nm against time. In this experiment you will usecommercially available dried yeast to produce a standard curve ofabsorbance at 440 nm against concentration (dry mass in grams/litre).You will then use your standard curve to determine the concentration ofyeast in each of the timed samples from the previous experiment anddraw a growth curve of cell concentration against time.



100 cm3 sterile yeast glucose broth in flask or bottle11 large test tubes (or universals)test-tube rackdried yeast (not fast acting)weighing boatspatula4 × 10 cm3 syringes (or pipettors with tips)1 cm3 pipettecuvettesdiscard jar containing 2% stericolgraph papermarker pen


balance (accurate to 0.01 g)colorimeter (440 nm)crushed ice (optional)


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Instructions

In order to make up the appropriate concentrations of yeast you willweigh out 0.2 g dried yeast and add it to 20 cm3 sterile broth to give aconcentration of 10 grams/litre. You will then use this suspension (thestandard dilution) to make further dilutions giving you yeastconcentrations in g/l of 5, 4, 3, 2.5, 2, 1.5, 1.0, 0.5, 0.25, 0.05. Look atthe table carefully and make sure you understand it before you start.

You may need crushed ice to keep your tubes cool while you carry outthe experiment.

1. Withdraw 20 cm3 broth from a flask of sterile yeast glucose brothand add to a test tube (or universal).

2. Add 0.2 g dried yeast to the broth in the test tube. This will giveyou a concentration of 10 g/litre. Shake gently occasionally untilthe cells are fully resuspended. This is your standard dilution. Useit to make further dilutions.

3. Label clean test tubes 1–10. Add the volume of sterile broth shownin Row A in the table on page 80 to each tube. Pay close attentionas you are required to add different volumes to the tubes.

4. Add the volumes from your standard dilution to tubes 1–5 shownin Row B.

Add the volumes from the numbered tubes shown in Row B totubes 6–10.

Note: Pay careful attention to tube numbers and volumes.Remember to suspend cells by shaking gently before taking asample and use a fresh syringe for each tube.

Dispose of used syringes and pipettes in the discard jar.

Make sure that you understand how the yeast concentration in g/lis worked out.


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Tube no 1 2 3 4 5 6 7 8 9 10

A volume 5 6 7 7.5 8 5 5 5 5 9

sterile

broth

(cm3)

B volume 5 4 3 2.5 2 5 cm3 5 cm3 5 cm3 5 cm3 1 cm3

yeast from from from from from

suspension tube 3 tube 5 tube 7 tube 8 tube 8

(cm3)

yeast conc. 5 4 3 2.5 2 1.5 1 0.5 0.25 0.05

(g/l)

5. Using sterile broth as the reference, calibrate the colorimeter (i.e.set it to zero) at 440 nm.

6. Starting with tube 10 shake the test tube to distribute the cellsevenly and use a pipette to transfer about 3 cm3 into a cuvette.

7. Measure the absorbance at 440 nm of the sample you have justwithdrawn.

8. Return the sample to its original tube and repeat steps 6 and 7,using the same pipette and cuvette, to obtain readings for tubes

9–1.

9. Draw a graph of your results with suitable scales and axes. This isthe standard curve which you can use to determine yeastconcentration from absorbances of the batch culture from theGrowth curve: determination of doubling time and growth rateconstant experiment.

10. Present your results from the experiment Growth curve:determination of doubling time and growth rate constant usingthe standard curve data.


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Using the standard curve to determine concentration in grams/litrefrom known absorbance

1. Use the absorbances measured in the previous experiment.

2. Draw a table showing time of sample, absorbance andconcentration.

3. Fill in the time and absorbance rows.

4. Carry out the following for each absorbance:

• Mark the value on the absorbance (vertical) axis of yourstandard curve.

• Draw a horizontal line till it meets the standard curve (a). Markthe point.

• Draw a vertical line from there to meet the yeast concentrationaxis (b).

• Read the value and complete the concentration row on thetable.

5. Plot a graph of yeast concentration against time.

6. Write a report on your practical placing particular emphasis onevaluation of the equipment and methods used with respect to theaccuracy and reliability of the results.

IncreasingAbsorbanceat 440nm

Increasing Yeast concentration (dry mass g/l)

(a)

(b)

Standard curve

Increasingabsorbanceat 440 nm

Increasing yeast concentration (dry mass g/l)


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ACTIVITY 11

Unit: Biotechnology (AH): Food Industry: Yeast extracts

Title: Viability of yeast at different stages of autolysis



This experiment can be used to:• provide evidence for the assessment of Outcome 3

• develop knowledge and understanding of the use of yeast biomassand the effect of autolysis treatments on yeast

• develop problem solving skills and in particular Outcome 2performance criteria:(b) information is accurately processed using calculations whereappropriate(c) conclusions drawn are valid and explanations given are supportedby evidence(d) experimental procedures are planned, designed and evaluatedappropriately.


In the food industry, yeast is grown in huge fermenters and then givendifferent treatments to produce different flavourings. All of thesetreatments involve the process of autolysis. This process of autolysisinvolves killing the yeast and encouraging the breakdown of the cells byenzymes. It is the products of enzyme degradation which produce thespecific flavour molecules. Autolysis usually begins with the addition ofsalt to the cells, causing water to leave the cells by osmosis andbeginning the process of cell breakdown. The cells are then heated,encouraging further breakdown of the cells.

In this experiment different treatments are used to autolyse yeast cells.The viability of the autolysed yeast is then tested in three different ways:plating out on agar; testing for dehydrogenase activity; microscopicexamination with methylene blue stain.


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The experiment is carried out over several days. The yeast samplesrequire to be autolysed overnight before testing for viability. At thediscretion of the teacher/lecturer the students may also be required toprepare their own yeast agar and if so time has to be allowed forautoclaving the agar, letting it cool and setting and drying of the pouredagar plates.

If the 3 tests have to be carried out on different days the rehydratedyeast samples should be stored in the fridge until required.

Supply of materials



Specific advice for performance criteria b–fPC b: to include an outline of the procedures, for example:

preparation of the agar plates; autolysis of the yeast samples;rehydration of the yeast samples; plating out on agar; stainingcells with methylene blue and examination under themicroscope; testing for dehydrogenase activity.

PC c: a table of results with appropriate headings and units showingfor each treatment:(i) growth of yeast on agar(ii) colour changes of resazurin with time(iii) numbers of blue and colourless yeast cells.

PC d: for the dehydrogenase activity method, a graph of rate of colourchange is drawn with colours on the y-axis and time on thex-axis. For the methylene blue method, the percentage of livingcells has been calculated correctly.

PC e: a conclusion is made about the effect of the different treatmentson the yeast viability and the correlations between each methodof testing viability.


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• accuracy in weighing and measuring out chemicals• the effectiveness of techniques used• difficulties in taking measurements, e.g. colour change,

counting cells• prevention of cross-contamination between autolysis

treatments• discrepancies between the different methods for testing

viability.

Extension work

A project can be carried out by expanding this method to include agreater range of yeast autolysis treatments.

A variety of different protease enzymes are used in industry, within theautolysis process, to aid the production of different flavours; studentscould experiment with the addition of protease enzymes at differentstages in the process.

A low cost laboratory fermenter has been developed in collaborationwith Quest International. It is possible to compare the growth of freshand autolysed yeast using this fermenter. In addition it is possible tomonitor the progress of the yeast as it goes through the process ofautolysis.

References

Berry, D. (1999), ‘Yeast Matters’, Biologist 46 (5) 211–214

Acknowledgment



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Technical guide


Yeast autolysis


10 g fresh yeast6 g salt5 × 200 cm3 beakersstirring rod


incubators at different temperatures, e.g. 40°C, 60°C, 80°Cbalance (accurate to 0.5 g)

Making the agar


100 cm3 beakerstirring rod3 McCartney bottles*weighing boat1 g glucose*1 g bactopeptone*0.5 g yeast extract*0.1 M sulphuric acid0.1 M sodium hydroxidedistilled watermarker pen10 cm3 syringe1 g agar*


* scale up quantities if molten agar/agar plates are being suppliedbalances (accurate to 0.5 g)pH probes or pH paperautoclave


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Pouring the agar


Ampholytic surfactant disinfectant and cloth for swabbing surfaceslabels3 sterile petri dishesBunsen burner3 McCartney bottles with sterile molten yeast agar (prepared by students

earlier)

Streaking an agar plate


3 yeast agar plates (prepared by students earlier)inoculating loop or 5 sterile plastic inoculating loopsrehydrated yeast samples (prepared by students)marker penSellotape


incubator at 30°C

Dehydrogenase enzyme activity


rehydrated yeast samples (prepared by students)test-tube rack with 5 test tubes5 labelsstop clock10 cm3 syringe/measuring cylinder20 cm3 resazurin dye20 cm3 5% glucose solutioncolour chart (a colour chart can be made from a paint colour chart of

appropriate hues)

Materials to be shared

waterbath at 35°C


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Testing yeast samples with methylene blue


rehydrated yeast samples (prepared by students)microscope (×400 magnification)microscope slides and coverslipsdropper0.1% methylene blue solution50 cm3 beakerstirring rod


Fresh yeast can be bought in large blocks and then cut intoapproximately 2 g pieces, covered in tin foil and then frozen untilrequired. Defrost thoroughly before use.

Bactopeptone can be purchased from: Becton Dickson, Micro systems,Cowley, Oxfordshire OX4 3LY. Tel: 01865 748844 Fax: 01865 781627.Cost £20.89 for 100 g (2000 prices).

Plastic inoculating can be purchased from: J. Wood, 39 Back SneddonStreet, Paisley. Tel: 0141 887 3531. Cost £32.00 for 1000 in packs of 20(2000 prices).

Resazurin tablets can be purchased from Philip Harris Education.Catalogue Number: S725103. Cost £5.15 for 25 tabs (2000 prices).One tablet is dissolved in 50 cm3 of water – make up immediately beforeuse.

Supply of materials



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Used inoculating loops should be disinfected and then disposed of withnormal waste.

Used agar plates should be autoclaved and then disposed of with normalwaste.


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Are you aware of the precautions you must take to preventcontamination of the agar plates?

Can you successfully examine material under a microscope at ×400magnification?

In your group decide how the activity will be managed by allocatingtasks to each member.

Recording of data

Prepare summary tables to record your group results.


Evaluation

How effective were the tests which you used?


Were there any possible sources of error?


What is the biological importance of the process which you areinvestigating?


BIOTECHNOLOGY


Introduction

Yeasts are versatile micro-organisms which have been used for centuriesto produce bread and alcoholic drinks.

In more recent years they have been used to produce flavourings for thefood industry. The yeast is grown in huge fermenters to producebiomass and then it is treated in different ways to produce differentflavourings. These flavourings are found in most of the savoury snackfoods that we eat – crisps, soups, snacks etc.

The yeast goes through a series of different treatments to develop thehuge variety of different end products. All of these treatments involvethe process of autolysis. The yeast products may be powders, granulesor pastes and they are then incorporated into processed foods toprovide natural flavourings.

This process of autolysis (auto: self; lysis: splitting) involves killing theyeast and encouraging the breakdown of the cells by enzymes. Thesemay be the cell’s own enzymes or enzymes may be added. It is theseproducts of enzyme degradation which produce the specific flavourmolecules. Autolysis usually begins with the addition of salt to the cells,causing water to leave the cells by osmosis and beginning the process ofcell breakdown. The cells are then heated, encouraging furtherbreakdown of the cells.

In this practical you are going to carry out the process of autolysis andtry to find at what point in the process the cells actually die. You willadd salt and heat yeast and then test the viability by plating out thetreated yeast to see if it will grow. You will also test the autolysedproduct to see if dehydrogenase enzymes are active and examine thecells microscopically to see if they take up methylene blue dye.

Yeast autolysis



10 g fresh yeastsalt5 × 200 cm3 beakersstirring rod


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ovens at different temperatures

Instructions

1. Autolyse the yeast in 4 different ways. You could alter thetemperature at which the yeast is autolysed or the amount of saltwhich is added to the yeast. One possible regime is suggestedbelow:

• 2 g yeast 1 g salt at room temperature overnight• 2 g yeast 1 g salt at 40°C overnight• 2 g yeast 1 g salt at 60°C overnight• 2 g yeast 1 g salt at 80°C overnight.

2. Collect dried yeast samples and rehydrate by slowly adding waterto the dried sample, stirring constantly. Continue to add water tothe samples to make them up to 100 cm3.

3. Autolyse a fresh sample of yeast. Mix 2 g yeast with 1 g salt and addwater to make this sample up to 100 cm3.

You can now test the viability of these 5 yeast samples by the 3 differentmethods below.

Method 1: Plating out autolysed yeast samples

If you are making your own agar plates, follow the methods below formaking and pouring agar. If your agar plates will be supplied, go to themethod for streaking an agar plate.

Making the agar – (this method will make enough agar for 3 petridishes)



100 cm3 beakerstirring rod3 McCartney bottlesweighing boatglucose


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bactopeptoneyeast extract0.1 M sulphuric acid0.1 M sodium hydroxidedistilled watermarker pen10 ml syringeagar

Materials to be shared:balances (accurate to 0.5 g)pH probes or pH paperautoclave

Instructions

1. Weigh out 1 g of glucose, 1 g of bactopeptone and 0.5 g of yeastextract.

2. Place the chemicals together in a beaker and add enough distilledwater to make up to the 50 cm3 mark on the beaker.

3. Stir thoroughly and then test the pH. Add enough sodiumhydroxide or sulphuric acid one drop at a time to make thesolution pH6 – test with pH probe or pH paper.

4. Place 15 cm3 of this solution into a McCartney bottle – you shouldhave enough for 3 bottles.

5. Add 0.3 g of agar to each McCartney bottle.

6. Shake and then place each lid loosely on the bottle and initial yourbottles.

Now give your bottles to your teacher/lecturer so that they can beautoclaved for 15 minutes.


BIOTECHNOLOGY

Pouring the agar



3 McCartney bottles with sterile molten yeast agardisinfectant and clothlabels3 sterile petri dishesBunsen burner

Instructions

1. Wash your hands.

2. Clean the area which you are going to work in with a cloth ortissue soaked in disinfectant.

3. You will be given 3 sterile petri dishes. Do not open them.

4. Collect 3 labels and write your initials, yeast agar and today’s dateon them.

5. Put a label onto the base of each petri dish (the base is smaller thanthe lid).

6. When the agar is cool enough to handle take off the lid of a bottleand flame the bottle in a cool Bunsen flame.

7. Pour the agar into the petri dish. Open the dish as little as you can.

8. Repeat for the other 2 petri dishes

9. Once the agar is set it is ready to be inoculated with the yeast.


BIOTECHNOLOGY

Streaking an agar plate



disinfectant and cloth3 yeast agar platesinoculating loop or 5 sterile plastic inoculating loopsrehydrated yeast samplesmarker penSellotape


incubator at 30°C

Instructions

1. Make sure that you are working in an area which has been swabbedwith disinfectant.

2. Turn your petri dish upside down and use a pen to label it with theyeast samples to be used, the date and your initials.

3. Open your petri dish and, using a sterile inoculating loop, streakyour sample of yeast onto one side of the petri dish.

4. Repeat with other yeast samples and plates.

5. Seal your plates with Sellotape and place them in an incubator at30°C for 3 days.

6. Record any growth of yeast over the next 5 days.


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Method 2: Comparing the activity of the dehydrogenase enzymespresent in autolysed yeast samples


During a metabolic pathway such as aerobic respiration, glucose isgradually broken down and energy is released. Hydrogen is releasedfrom the glucose in a process known as oxidation. This hydrogen bindsto a co-enzyme and each reaction is catalysed by an enzyme known as adehydrogenase.

Although it would not be possible to detect this reaction in a test tubesome chemicals such as resazurin dye change colour when they gainhydrogen.

It changes colour in the following ways:

blue → lilac → mauve → pink → colourless (unreduced) (partially reduced) (reduced)

You can use this reaction to compare the activity of the dehydrogenaseenzymes present in your 5 different autolysed yeast samples.

The time it takes for the dye to change colour will indicate the activity –the faster the colour change takes place the greater the activity of thedehydrogenase enzymes. Activity of enzymes such as dehydrogenaseswould indicate that the yeast is likely to be viable.



rehydrated yeast samplestest-tube rack with 5 test tubes5 labelsstop clockpair of safety spectaclessyringe/measuring cylinder20 cm3 resazurin dye20 cm3 5% glucose solutioncolour chart


waterbath at 35°C


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Instructions

1. Collect the materials listed on page 96.

2. Label 5 test tubes and add 3 cm3 of resazurin dye to each tube.

3. Add 3 cm3 of yeast suspension to each tube.

4. Shake each tube and place in a waterbath at 35°C.

5. Using the colour chart, record the colour of each tube every2 minutes for 20 minutes.

If you do not get a reaction in any of the tubes after 10 minutesadd 3 cm3 of 5 % glucose solution to each test tube and shake.

The chart below shows the colour changes which you might expectand the order in which they will appear.

1 2 3 4 5Blue Lilac Mauve Pink Colourless

6. Record the results in a table with suitable headings.

7. Present your results as a graph with suitable scales and axes.


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Method 3: Testing yeast samples with methylene blue

Methylene blue dye will diffuse into the yeast cells. If the cells are livingthey will pump the blue dye out but if they are dead they will remainblue.



rehydrated yeast samplesmicroscopemicroscope slides and coverslipsdropper0.1% methylene blue solution50 cm3 beakerstirring roddistilled water

Instructions

1. Place a drop of the diluted yeast onto the microscope slide andadd a drop of methylene blue dye and wait 5 minutes.

2. Place the slide onto the stage of your microscope and focus.

3. Count all the blue cells and clear cells in your field of view.

You may find that there are too many cells to count. If this is thecase then you can dilute your samples and start again.

4. Calculate the percentage of viable cells.

5. Record your results in a suitable table.


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ACTIVITY 12

Unit: Biotechnology (AH): Food Industry: Fruit juice production

Title: Fruit juice production



This experiment can be used to:• provide evidence for the assessment of Outcome 3• develop knowledge and understanding of the use of cellulases,

pectinases and amylases in the production of fruit drinks• develop problem solving skills and in particular Outcome 2

performance criteria:(c) conclusions drawn are valid and explanations given are

supported by evidence(d) experimental procedures are planned, designed and evaluated

appropriately.


Fruit juice companies use a variety of different treatments and enzymesto maximise their yield. The enzymes which are used are designed tobreak down the cell walls within the fruits and release the liquid andsugars which make up the fruit. Pectinases, amylases and cellulases allbreak down different structures of fruit cells and so affect the extractionprocess in different ways. During breakdown of fruit cells a variety ofpolysaccharides are found within the juice extract. These can cause thejuice to become cloudy and reduce its market value. Pectinases andamylases can both break down these insoluble compounds, releasingsoluble sugars that clarify the juice to produce a clearer, sweeterproduct.

In this experiment students will investigate the effectiveness of thesedifferent enzymes. They should be able to draw conclusions about theuse these enzymes could be put to in commercial fruit juice production.

FURTHER PR ACTICAL ACTIVITIES (AH BIOLOGY)1 0 0

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The experiment will take about 1 hour to complete.

The experiment can be developed further by using the SupplementaryStudent Information Sheet to investigate different ways of re-using theenzymes.

Supply of materials



Specific advice for performance criteria b–fPC b: to include the preparation of the apple; method of extracting

juice; methods for measuring fruit juice quantity and quality.

PC c: a table of results with appropriate headings and units showingthe type of enzyme treatment; volume of juice collected againsttime; clarity of the juice extracted; weight of waste apple.

PC d: a graph of rate of juice production with volume of juiceextracted on the y-axis and time on the x-axis.

PC e: a conclusion is drawn, using the qualitative and quantitativeresults, as to the effectiveness of the enzymes in extracting juice.


• accuracy in taking measurements• apple preparation (variety of apple, age of apple, how apple

has been preserved, size of apple pieces)• viscosity of enzymes• possible losses of juice during the extraction process (e.g. on

to filter paper if not dampened)• consistency of the enzyme coatings on the apple pieces• definition of effectiveness of extraction with relation to

quality (e.g. clarity) as well as quantity.

FURTHER PRACTICAL ACTIVITIES (AH BIOLOGY) 1 0 1

BIOTECHNOLOGY

Extension work

Different fruits and vegetables can be used.

The enzyme data sheets can be used to identify the optimum pH andtemperature of these enzymes and this can be tested throughexperimentation.

The enzymes can be immobilised and continual re-use of the enzymescan be tested (see supplementary sheet).

Different methods can be used to investigate re-using the enzymes (seesupplementary sheet).

A comparison can be made between these enzymes and other amylases,pectinases and cellulases.

Find out the optimum combination of enzymes for maximum yield.

Determine the effect that reducing the concentration of enzyme has onyield.

References

NCBE booklet ‘In a jam and out of juice’.Available free from NCBE’s website: http://www.ncbe.reading.ac.ukor from NCBE, The University of Reading, Whiteknights, PO Box 228,Reading RG6 6AJ. Tel: 0118 987 3743. Fax: 0118 975 0140.

Acknowledgement



BIOTECHNOLOGY

Technical guide

Materials required


apple/apple sauceknifeTermamyl/Pectinex/Celluclast4 × 10 cm3 measuring cylindersfilter papers4 small filter funnelstimer4 × 100 cm3 beakers4 weigh boatsstirring rod5 cuvettes (or test tubes if suitable colorimeter is used)


balance (accurate to 0.1 g)colorimeter


An ordinary eating apple should be used or tins of apple sauce.

Plastic measuring cylinders and filter funnels are best for this activity.

Termamyl/Pectinex/Celluclast can be obtained from the National Centrefor Biotechnology Education (NCBE), The University of Reading,Whiteknights, PO Box 228, Reading RG6 6AJ. Tel: 0118 9873743.Enzyme data sheets are automatically supplied with the order.

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.

Supply of materials

It 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. Trays


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could be provided containing one type of specialist equipment ormaterials.

For the supplementary part of this activity in addition to the abovematerials:


Enzyme Immobilisation Instruction Cards200 cm3 1.5% calcium chloride10 cm3 4% sodium alginateenzyme solution, e.g. Pectinex/Celluclastdistilled water250 cm3 beaker10 cm3 syringe20 cm3 syringetea straineriron fillingsbar magnet in a plastic bag


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Collect a data sheet for the enzymes Termamyl, Pectinex and Celluclastand familiarise yourself with the action and commercial use of theseenzymes.

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 data

Prepare tables to record your group results.


Evaluation

How effective were the tests which you used?




What factors other than the enzyme used might affect the volume ofjuice produced?


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What factors, other than volume of juice produced, might be importantin determining effectiveness of the procedures?

What ideas do you have for further work?

What is the economic importance of the process that you are studying?


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Introduction

Fruit juice can be extracted from a wide variety of fruits. This can bedone by simply squeezing the fruits but fruit juice companies useenzymes to increase the volume of juice produced and the speed ofextraction. The enzymes which are used are designed to break downthe cell walls within the fruits and release the liquid and sugars whichmake up the fruit. Fruit cell walls are very complex molecular structuresand to get the maximum breakdown of the compounds found in themfruit juice companies use a variety of different treatments and enzymesto maximise their yield. Fruit is made up of cells linked by middlelamellae which contain insoluble proto-pectin. Pectinase breaks downthe pectin chains and therefore reduces its binding action. The cellwalls are composed largely of cellulose and hemicellulose and cellulasesweaken the cell walls and make it easier to extract the juice. As thebreakdown of the fruit cells continues a variety of polysaccharides arefound within the juice extract; these can cause the juice to becomecloudy and reduce its market value. Pectinases and amylases can bothbreak down these insoluble compounds, releasing soluble sugars thatclarify the juice to produce a clearer, sweeter product. Differentcombinations of enzymes are used with each different type of fruit.

Enzymes are expensive products and clearly juice manufacturers wouldwish to minimise their costs by using the enzymes at their optimumconditions, and therefore maximising their effectiveness, and re-usingthe enzymes where possible.

You are going to set out an investigation to look at the effectiveness ofthese different enzymes.



appleknifeTermamyl/Pectinex/Celluclast4 × 10 cm3 measuring cylindersfilter paper4 small filter funnelstimer4 × 100 cm3 beakers4 weigh boats


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stirring rod5 cuvettes (or test tubes)


balancecolorimeter

Instructions

1. Cut up the apple into small pieces.

2. Divide up the chopped apple into roughly 4 equal parts and usethe balance to weigh each part to equalise accurately and recordthe weight.

3. Place each quarter into a beaker and add 2 cm3 of Teramyl to onebeaker, 2 cm3 of Pectinex to another, 2 cm3 of Celluclast to a thirdand 2 cm3 of distilled water to the last beaker.

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.

4. Stir each beaker and leave for 5 minutes.

5. Place beaker contents into a funnel and filter the apple juice intothe measuring cylinders.

6. Record the volume of juice every 2 minutes and the final volume ofjuice.

7. Take a sample of each juice and take a measure its clarity by using acolorimeter.

8. Weigh the waste apple from each extraction.

9. Record your results in a table with suitable headings.

10. Calculate the yield of fruit juice.

11. Graph your results to show the rate of fruit extraction.


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Re-using enzymes

IntroductionYou are going to try immobilising the cellulase or the pectinaseenzymes, or a combination of the two, to see if you can extract theenzymes from the waste apple and re-use them.

You will probably already be familiar with the technique of enzymeimmobilisation. If you cannot remember how to immobilise enzymescollect an Enzyme Immobilisation Instruction Card.

Ideally you should work in a group of about 4 people and share out the4 tasks shown in the table on page 109.

Additional materials required:

1.5% calcium chloride4% sodium alginatedistilled water250 cm3 beaker10 cm3 syringe20 cm3 syringetea straineriron fillingsbar magnet in a plastic bag


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Enzyme/s• Immobilise the enzyme/s• Use the immobilised beads to

extract juice and then removethe beads from the waste fruitand re-use them. Compareyour results with the resultsfrom the initial experiment.

Enzyme/s plus air bubbles• Immobilise the enzyme/s using

a 20 cm3 syringe, adding airbubbles to the enzyme/alginatemixture by passing it in andout of the syringe beforeplacing into calcium chloride

• Use the immobilised beads toextract the juice from the fruit

• Place the waste apple into abeaker of water and see if thebeads float and are easier toremove from the waste fruit

• Re-use the beads. Compareyour results with the resultsfrom the initial experiment.

Enzyme/s and iron fillings• Add 3 g of iron fillings to the

enzyme/s• Immobilise the enzyme/s and

iron fillings• Use the immobilised beads to

extract the juice from the fruit• Place the waste apple into a

beaker of water and use amagnet to try to remove thebeads from the apple


Enzyme/s plus air bubbles andiron fillings• Add 3 g of iron fillings to the

enzyme/s• Immobilise the enzyme/s and

iron fillings using a 20 cm3

syringe, adding air bubbles tothe enzyme/alginate mixtureby passing it in and out of thesyringe before placing intocalcium chloride

• Use the immobilised beads toextract the juice from the fruit

• Place the waste apple into abeaker of water and use amagnet to try to remove thebeads from the apple


Now work as a team to discuss the results of the 4 different experimentsand further work which could be done to find out more about theaction of these enzymes.


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Enzyme immobilisation instruction card

IntroductionEnzymes such as pectinase are often used in industrial processes. At theend of the process the enzyme is often mixed up with the product andcannot be easily separated from it. Immobilisation is a method whichtraps the enzymes in a bead which can be more easily separated fromthe product. The method also means that it may be possible to re-usethe enzyme once it has been separated from the product. This methodis often used in a continuous flow system.


4% sodium alginate1.5% calcium chlorideenzyme solution, e.g. pectinasedistilled water250 cm3 beaker10 cm3 syringetea strainer

InstructionsThe enzyme is mixed with sodium alginate and then fixed in a solutionof calcium chloride.

1. Draw up 3 cm3 of enzyme solution into a syringe (if you are using acombination treatment use 1.5 cm3 of each enzyme).

2. Add 7 cm3 of sodium alginate to the same syringe and mixthoroughly in the syringe.

3. Gently, drop by drop, release the contents of the syringe to abeaker containing calcium chloride solution and leave for 5minutes to set.

4. Place the beads in a small tea strainer and thoroughly rinse them indistilled water.

5. Now place the beads in the apple puree, leave for 5 minutes andthen place in the filter funnel.


ANIMAL BEHAVIOUR

ACTIVITY 13

Unit: Animal Behaviour (AH): Recording and interpretation ofBehaviour

Title: Turn alteration in woodlice

Teacher/lecturer/technical guide

Introduction

This practical is taken from Animal Behaviour – Practical work anddata response exercises for sixth form students by Michael Dockery andMichael Reiss, published by the Association for the Study of AnimalBehaviour (1996). This book is available from Michael Dockery,Department of Biological Sciences, John Dalton Building, ManchesterMetropolitan University, Chester Street, Manchester, M1 5GD (Tel: 0161247 6365; E-mail: [email protected]). This book contains all theinformation necessary for a teacher/lecturer and a technical guide.


Specific advice for performance criteria b–fPC b: to include: description/diagram of maze; order of forced turns

and outline of procedure.

PC c: a table of results with appropriate headings showing thenumbers of turn preferences for each forced turn.

PC d: a statistical analysis is carried out (chi-squared test of associationor binomial test) to ascertain if turn alteration has occurred.

PC e: conclusion related to turn alteration hypothesis.


ANIMAL BEHAVIOUR

PC f: evaluation points include:• number of woodlice used• value of statistical analysis• different species of woodlice• possibility of a chemical trail left by woodlice• ensuring each woodlouse is used only once• the order of forced turns.


ANIMAL BEHAVIOUR





What hypothesis is being tested?



What measurements/observations are you going to make?


What precautions are required when handling woodlice?

In your groups decide how the activity will be managed by allocatingtasks to each member.

Consult a book on statistics or consult your teacher/lecturer on usingrandom number tables and statistical tests.

Recording of data

Prepare a table to record the results. You should use a ruler, correctheadings and appropriate units.

Evaluation

Were all the woodlice of the same species?

How many woodlice should be used?

Why was a statistical analysis used?

Why was the floor of the maze washed between woodlice?

What was the significance of the order of forced turns?


ANIMAL BEHAVIOUR


Introduction

A number of research studies have shown that in some species ofvertebrates turn alteration, or correcting behaviour, occurs. Turnalteration is seen when an organism tends to turn in the oppositedirection to a prior forced turn. Thus if an animal is in a simple mazeand is forced to turn right, it will turn left at the next choice point in themaze, or at least show a preference for turning left. In this practical youcan see if woodlice show turn alteration.

This practical lends itself to simple statistical analysis of the results to seeif the measured differences between experimental groups are significant.Binomial or chi-squared tests are suitable.



H maze20–30 woodlicecontainer with moist paper towels/damp soil to keep woodliceartist’s brush to handle woodlicecotton wool swabsde-ionised waterstatistical tables

Woodlice have delicate legs that can be damaged so transfer themcarefully using an artist’s brush. Keep them in a damp container andallow them to settle before the first trial. Keep the wodlice in thelaboratory for as short a time as possible and return them to theirnatural habitat as soon as possible.

Instructions

1. Determine the order you will force them to turn right or left. Youmay wish to do this alternately or use random number tables froma book of statistical tables to determine the order of forced turns.Roughly half should be forced to turn right, the other half left.

2. Select the direction of the forced turn and place one of thewoodlice in the maze using the artist’s brush.


ANIMAL BEHAVIOUR

3. When the woodlouse leaves the choice point record whether itturned right or left.

4. Remove the woodlouse from the maze and ensure it cannot beused again.

5. Using a cotton wool swab moistened with de-ionised water, washthe floor of the maze.

6. Repeat the procedure with the remaining woodlice, changing theforced turn as appropriate.

7. Present your results in a suitable table with the results of astatistical analysis.


ANIMAL BEHAVIOUR

ACTIVITY 14

Unit: Animal Behaviour (AH): Innate and learned behaviour

Title: Maze learning in small mammals


Introduction

This practical is taken from Animal Behaviour – Practical work anddata response exercises for sixth form students by Michael Dockery andMichael Reiss, published by the Association for the Study of AnimalBehaviour (1996). This book is available from Michael Dockery,Department of Biological Sciences, John Dalton Building, ManchesterMetropolitan University, Chester Street, Manchester, M1 5GD (Tel: 0161247 6365; E-mail: [email protected]). This book contains all theinformation necessary for a teacher/lecturer and a technical guide.

This practical can be used with several animals of one species or withmore than one species in a comparative study.


Specific advice for performance criteria b–fPC b: to include: description/diagram of maze; outline of procedure

and note of animals/species used.

PC c: results are recorded in a table with suitable headings showingtime taken and errors made for each trial.

PC d: graphs of time taken against trial number and errors madeagainst trial number for individuals or species.

PC e: comparisons are made between different species/individuals andcomments made on both time taken and errors made.

PC f: evaluation points include:• animal should be hungry, to provide motivation• comment on errors and time as measure of performance• washing maze to remove possibility of chemical trails.


ANIMAL BEHAVIOUR









What precautions are required when handling small mammals?


Recording of data


Evaluation

Why should the animal not have eaten large amounts of food prior tothe practical?

Why measure both errors made and time taken in completing the maze?

Why was the maze washed between animals?


ANIMAL BEHAVIOUR


Introduction

Behaviour has both genetically and environmentally determinedcomponents. Although learning is mainly an environmentallydetermined influence, it is not possible to separate it entirely frominnate or genetically determined influence. Many animals can learn tofind their way through a maze, especially if they are given a reward onsuccessfully completing their journey. In this practical you can see howsmall mammals learn their way through a maze.


Materials required by each student/group:maze suitable for small mammalsdifferent species of small mammals (e.g. gerbils, hamsters, mice)stopwatchnon-smelly food (e.g small nut or food pellet)

Small mammals that are used to being handled are less likely to find thisexercise stressful. The animal should not have eaten large amounts offood prior to the practical. (However, they should not be undulydeprived of food for the purposes of the practical.) The area where thepractical is to take place should be quiet and calm. You should followlocal guidelines when handling and caring for the animals.

Instructions

1. Wash and dry the maze with warm, non-soapy water.

2. Place the animal in the starting area without there being any foodin the food area.

3. Let the animal find its way to the food area.

4. Time how long this takes and score the number of errors it makeson the way.

5. When the animal arrives at the food area, give it a small item offood. Once this has been eaten, repeat steps 2, 3 and 4 until theanimal has learned to complete the maze 3 times in a row withoutmaking any errors or you have conducted 10 trials (whichevercomes first).


ANIMAL BEHAVIOUR


7. Wash and dry the maze and repeat the process with anotheranimal.

8. Present your results as a graph with suitable scales and axeslabelled with quantities and units.


ANIMAL BEHAVIOUR

ACTIVITY 15

Unit: Animal Behaviour (AH): Feeding behaviour

Title: Vigilance behaviour in barnacle geese


Introduction

This practical is based on the video Vigilance Behaviour in BarnacleGeese and its associated notes produced by the Association for the Studyof Animal Behaviour. The video and its accompanying notes areavailable from Michael Dockery, Department of Biological Sciences, JohnDalton Building, Manchester Metropolitan University, Chester Street,Manchester, M1 5GD (Tel: 0161 247 6365; E-mail:[email protected]). The accompanying notes contain all theinformation necessary for a teacher/lecturer and a technical guide.


Specific advice for performance criteria b–fPC b: to include: counting number of ‘head ups’; timing of each ‘head

up’ and multiplying to calculate total time vigilant for each flocksize.

PC c: results are recorded in a table with suitable headings showingflock size, number of ‘head ups’, time of each head up and totaltime vigilant.

PC d: total time vigilant for each flock size is calculated correctly.Graph of time vigilant against flock size is made. (Graph ofsurveillance, i.e. number of ‘head ups’ against flock size couldalso be made although not strictly necessary.)

PC e: time vigilant decreases with flock size, allowing more feedingtime as vigilance duties are shared in the flock. Surveillance rate(i.e. number of ‘head ups’) does not vary significantly with flocksize.


ANIMAL BEHAVIOUR

PC f: evaluation points include:• the importance of studying stable undisturbed flocks• factors other than group size that might affect vigilance, such

as: age of birds, familiarity with environment, position in flock(periphery or centre, for example).


ANIMAL BEHAVIOUR









How will you calculate total time vigilant?


Recording of data


Evaluation

What other factors may affect vigilance behaviour?

What are the pros and cons of feeding in a group?

Why were only undisturbed stable flocks filmed?


ANIMAL BEHAVIOUR


Introduction

Barnacle geese come to the UK in winter from their breeding grounds inGreenland to feed on the rich grasslands and estuaries along ourcoastline. Seventy per cent (18 000 birds) of Greenland’s barnacle geeseoverwinter on the RSPB reserve at Loch Gruinart on Islay where thisvideo footage was shot.

When an individual is foraging for food it has to balance the risk offeeding with the risk of possible predation. Gathering in flocks to feedgives an earlier warning of the approach of a suspected predator.Barnacle geese have classic feeding and vigilant postures making themexcellent subjects for a study of vigilance. When feeding their heads aredown and when vigilant they adopt a ‘head up’ posture.

The video you are about to watch after an introductory sequence shows21 sequences of the vigilance behaviour of barnacle geese in flocks ofdifferent sizes.


Stopwatch for each studentVideo

Instructions

1. For each sequence record the flock size, number of ‘head ups’ andtime of each ‘head up’.

2. From your data calculate the total time vigilant.



PHYSIOLOGY, HEALTH AND EXERCISE

ACTIVITY 16

Unit: Physiology, Health and Exercise (AH): Exercise and thecardiovascular system

Title: The effects of exercise on heart rate and blood pressure




• provide evidence for assessment of Outcome 3• develop knowledge and understanding of the effect of exercise on

the cardiovascular system• develop problem solving skills and in particular Outcome 2

performance criterion:(d) experimental procedures are planned, designed and evaluated

appropriately.


The guides have been written up for measurement of heart rate andblood pressure before and after varying degrees of exercise. Recoveryrates could also be measured and individuals with varying lifestyles/degrees of fitness could be investigated. These measurements couldprovide alternative experiments to this one to provide evidence forOutcome 3 or be combined into a longer study for the BiologyInvestigation unit. Heart rate can be determined with a pulse monitor,or by hand (practice required), blood pressure with asphygmomanometer.

Measurements are recorded at rest, after gentle exercise and aftervigorous exercise.

Suitable exercises could include: step-upsjogging on spotexercise bike, etc.




Health and safety of students should be of prime consideration.Guidance on students as subjects of experiment or investigation shouldbe sought from employers. A typical sample of such guidance isprovided in the technical guide. Students who cannot be subjects of theexperiment can take on active roles such as measuring pulse rates orrecording results. Students should work in groups and should ultimatelygain a record of results for one or more members of their group. Inpreparing for the activity, students should decide in groups on theselection and duration of exercises as well as how the results are to bemeasured and collected. It may be helpful for them to construct a flowchart of the procedure they are to follow (this could also be used laterin the method section of the write up).Class results can be pooled to look at overall trends.


Specific advice for performance criteria b–fPC b: Aim is to determine the effect of varying levels of exercise on

heart rate.

Methodology: What exercise was carried out?How were different levels of exercise achieved?Control measures to include: time each level ofexercise was performed; consistent method ofmeasuring pulse rate and blood pressure; howmeasurements were taken.

PC c: Tables of raw data to include: level of exercise, pulse rate (beatsper minute) and blood pressure.Class results could be pooled to give averages for heart rate andblood pressure.

PC d: Class results can be shown as bar graphs/line graphs asappropriate for heart rate, systolic and diastolic blood pressure.

PC e: Heart rate increases with increase in level of exercise. Systolicblood pressure may increase slightly with increase in level ofexercise and diastolic blood pressure is likely to remainrelatively unchanged.



PC f: Evaluation points could include:• effectiveness of procedures

– repeats required for reliability• control of variables

– individual differences between subjects (age/sex/fitnesslevels/health etc.)

• limitations of equipment– reliability of blood pressure monitors/pulse rate monitors

• possible sources of error– human error in operating measuring devices/manually

recording pulse rates– pulse rate dependent on other factors which cannot be

controlled (e.g. emotional state)• possible improvements

– anything that addresses any of the above in a positive way.



Technical guide

Materials required


1 pulse monitor (optional)1 stop clock1 sphygmomanometer (digital sphygmomanometers designed for the

home health/fitness market are suitable)1 step/exercise bike (optional)1 metronome – to adjust stepping rate (optional)

Students as subjects of experiment or investigation

Employers’ guidelines on students as subjects of experiment orinvestigation should be followed. The following guidelines are typical ofthose used by employers to avoid putting students into situations ofemotional or physical stress.

1. There should be no pressure on students to perform or take partin any experiment or activity that involves exercise or bodymeasurements of themselves.

2. Information should be obtained about the medical condition ofstudents before proceeding with an experiment. Students withmedical problems (epilepsy, asthma etc.) must not be put at risk.

3. Students who are excused PE activities on medical grounds shouldnot be the subject of investigations which examine the effects ofexercise on breathing or pulse rate.

4. The choice of physical exercise should be sufficient to demonstrateeffect without being excessive.

5. Only apparatus designed for educational or personal use should beused for physiological measurements. Students should be aware ofthe limitations of use of any such apparatus and teachers/lecturersshould emphasise that the use of apparatus for physiologicalmeasurements is educational and not diagnostic.

6. Sensitivity should be applied in the use of results. Names shouldnot be attributed to measurements.







Check availability of equipment and materials. Do you require anyalternatives?

Is there any equipment you are not familiar with? If so, ask your teacher/lecturer for help.

Do you know how to carry out each step? Do you require further helpor information from, for example, a Student Supplementary InformationSheet?



What variable is being studied?

What variables must be kept constant to ensure the validity of theresults?

What measurements/observations are you going to make? Whatequipment will you use to make these?

What is the control in the activity? Are further control measuresnecessary?

Recording of data

Construct a table for recording your data (measurements/observationsmade during the activity). You should use a ruler, correct units andsuitable headings.


In your groups 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.



Evaluation

Were there any variables that were difficult to control in yourexperiment?

Was the equipment reliable?





The measurement of the pulse at any appropriate position is a goodmeasurement of heart rate as each heart beat forces blood through thearteries creating a pulse.

During exercise, muscle tissue requires more oxygen and glucose tosupply the energy needed to work. During exercise there is anincreased blood flow to the exercising muscles, aided by vasodilation ofarterioles supplying the active muscles and vasoconstriction of arteriesto organs which are not active during exercise (e.g. the gut). Suchchanges in blood flow can affect blood pressure.


Materials required by each student/group:1 pulse monitor (optional)1 stop clock1 sphygmomanometer (digital sphygmomanometers designed for the

home health/fitness market are suitable)1 step/exercise bike (optional)1 metronome – to adjust stepping rate (optional)

Instructions

You are going to record two measurements during the course of thisexperiment:

a) pulse rateb) blood pressure

1. Practise measuring pulse rates and blood pressure with a partnerto familiarise yourself with the equipment and its instructions foruse.

Your teacher/lecturer may provide pulse monitors or you may haveto do this manually, using the radial artery at the wrist. It is usualto count the pulse over 20 seconds and then use this to work outthe pulse in one minute.

2. Record the pulse rate and blood pressure of the subject(s) at rest.



3. The subject should then carry out the selected exercise. Thisshould be low impact/gentle exercise (such as stepping on and offa step-up box for a period of three minutes).

4. Record the pulse rate and blood pressure of the subjectimmediately following this activity.

5. After a period of rest the subject should carry out a higher impact/more strenuous variation of the first activity by, for example,increasing the speed of stepping on and off the step-up box forthree minutes.

6. Record the pulse rate and blood pressure of the subjectimmediately following this activity.

7. Prepare suitable tables and record your results.

8. Pool the class results.

9. Use the class results to calculate the average heart rate and bloodpressure for each level of exercise.

10. Present the results in a graph with suitable scales and axes labelledwith quantities and units.

Further Student Activities

This investigation can be extended to enable the subjects’ physicalfitness to be estimated. Following a suitable exercise routine the pulserate should be recorded immediately after exercise and then at two-minute intervals until it returns to its pre-exercise level.

The post-exercise recovery time is the time taken for the pulse rate toreturn to its normal ‘resting ‘ level. The faster the recovery time themore physically fit the subject, assuming that they have exercised fully.



ACTIVITY 17

Unit: Physiology, Health and Exercise (AH): Exercise andmetabolism

Title: Energy expenditure for a day’s activities



This experiment can be used to:• provide evidence for assessment of Outcome 3• develop knowledge and understanding of basal metabolic rate and

energy needs• develop problem solving skills and in particular Outcome 2

performance criterion:(d) Experimental procedures are planned, designed and evaluated

appropriately.


The Basal Metabolic Rate (BMR) of an individual is the energy requiredto carry out normal body functions such as breathing and therefore mustalso be included in any calculation of energy expenditure for anindividual.

The total energy expenditure of an individual can be calculated if thereis an accurately timed record of all the activities of that individual and ifthe energy cost of each activity is known. A so-called activity diary,which covers all 1440 minutes of the day, should be kept. The energycost of each activity can be measured by indirect calorimetry, orestimated from published values. The Physical Activity Ratio (PAR) valueis the energy cost of an activity expressed as a multiple of BMR.


This investigation should not be carried out by students on a school daysince they have to continually record their activities.Students should be issued with either an activity diary or a log sheet torecord their raw data.



A basic set of energy values has been included in the studentsupplementary information, but teachers may wish their students tohave access to much fuller sets of data, such as those found in:

James, W. P. T. and Schofield (1990), Human Energy Requirements,Oxford University Press.McArdle W. O., Katch F. L. and Katch V. L. (1999), Sports and ExerciseNutrition, Lippincott, Williams and Wilkins.

Extension work

Use portable respirometers to make indirect calorimetry measurements.


Specific advice for performance criteria b–fPC b: Aim is to determine the daily energy expenditure of AH

students. Key points should include a summary of how the rawdata was obtained; monitoring changes in activity to includeaccurate timing of length of each activity.

PC c: Raw data showing the length of time spent on each differentactivity.

PC d: A table showing the above information and the PAR figure, BMRand energy expenditure for each activity.Calculation of overall energy expenditure from the table(could include histogram of activities or groups of activitieshere).

PC e: Conclusion related to daily energy expenditure.

PC f: Possible sources of errors:• difficulty of task means that errors will inevitably occur, e.g.

forgetting to log a change in activity. This will be reduced if‘helpers’ or other reminding strategies are employed.

• difficulty of matching up activities and published energyvalues means that some values will not be entirely accurate.

Possible improvements:• keep activity diary/log for longer to gain a more realistic

pattern of everyday life.



Technical guide

Materials required

1 set of weighing scalesrecord sheets and/or activity diarytables of PAR values

A basic table of PAR values is included as supplementary studentinformation. Further data on PAR values for a wider range of activitiescan be found in:

James, W. P. T. and Schofield (1990), Human Energy Requirements,Oxford University Press.McArdle W. O., Katch F. L. and Katch V. L. (1999), Sports and ExerciseNutrition, Lippincott, Williams and Wilkins.






Do you know how to carry out each step? Do you require further helpor information from, for example, a Student Supplementary InformationSheet?



What variables must be kept constant to ensure the validity of theresults?


Recording of data

Construct a table for recording your data (measurements/observationsmade during the activity). You should use a ruler, correct headings andappropriate units.

This investigation will be carried out on an individual basis but anopportunity will exist to collate the data obtained by some otherstudents. A commitment to this practical and to collecting the resultsfrom other students will be necessary to achieve Outcome 3.

Evaluation

How will you ensure every minute is accounted for?

Will the time scale of the experiment give an accurate reflection of yourdaily energy expenditure?





Your total energy expenditure in a 24-hour period is all the energy,measured in kilojoules, that your body uses up to carry out its dailyactivities. To calculate this figure it is necessary to keep an accuratetimed record of all your activities over 24 hours. You can choose to fillin an activity diary or a log sheet. Having gathered this information, itwill then be necessary to find out the energy cost of each activity. Forthe purpose of this investigation, you will use published values, but theenergy expended during activity can be measured by direct calorimetrymethods. The table of values will also provide you with a PhysicalActivity Ratio (PAR) value, which is the energy cost of an activityexpressed as a multiple of the Basic Metabolic Rate (BMR).

Calculation of energy expenditure also requires knowledge of your ownBMR. This is the energy expended, even at rest, to carry out normalbody functions, such as breathing. If you are resting the PAR value is 1.0,since this is assumed to be equivalent to BMR. Any form of physicalactivity is going to have a significant effect on energy expenditure. Themore physically demanding the activity, the greater the total energyexpenditure.


Materials required by each student/group:record sheets and/or activity diarytables of PAR values

Materials to be shared:1 set of weighing scales



Instructions

1. This investigation requires you to keep an accurate record of yourown activities over a 24-hour period. This record should be madeon either a Saturday or Sunday or other school closure day.

2. Your teacher/lecturer will provide you with either a logging sheetor an activity diary to enable you to keep an accurate timedrecord of your activities. This will provide you with the raw datathat you need.

3. Use an accurate set of scales to find your body weight. Use thisweight to calculate your BMR per hour. The supplementarystudent information sheet gives the appropriate figures for yourage group and provides a worked example.

4. Calculate energy expenditure by multiplying the duration of eachactivity by the appropriate PAR figure followed by the BMR/hour. (Atable of PAR values is included in the student information sheetalong with the worked example.)

5. Show your findings in an appropriate table.

6. Work out your total energy expenditure.

7. Collect results from other members of the class. Present theresults in a suitable format.




Sex BMR (MJ/day)

Male 0.063W + 2.896

Female 0.062W + 2.036

(W = body weight)

Jill is a student who weighs 60 kg. Her BMR is calculated as follows:(0.062 × 60) + 2.036 =

Table of typical PAR values

Category of activity Average PAR value

Sleeping / lying at rest 1.0

Quiet sitting activities (watching TV and

reading) 1.2

Active sitting activities (using computer and

driving) 1.6

Stationary standing activities (ironing) 1.4

Personal activities (washing, dressing and

eating) 1.4

Slow moving activities (cleaning, cooking and

bowling) 2.1

Walking 2.8

Physical activity (gardening) 3.7

Swimming, dancing and fast walking 4.8

Jogging, football and tennis 6.0



Physical activity log sheet

Activity Start time Finish time Duration



01

23

45

67

89

01

23

45

67

800

0004

0000

1004

1000

2004

2000

3004

3000

4004

4000

5004

5001

0005

0001

1005

1001

2005

2001

3005

3001

4005

4001

5005

5002

0006

0002

1006

1002

2006

2002

3006

3002

4006

4002

5006

5003

0007

0003

1007

1003

2007

2003

3007

3003

4007

4003

5007

50

Nam

e:

Dat

e:

Act

ivit

y d

iary

Use

sym

bo

ls t

o r

epre

sen

t yo

ur

acti

viti

es.

On

ly e

nte

r re

cord

s w

hen

yo

u s

tart

a n

ew a

ctiv

ity.



01

23

45

67

89

01

23

45

67

812

0016

0012

1016

1012

2016

2012

3016

3012

4016

4012

5016

5013

0017

0013

1017

1013

2017

2013

3017

3013

4017

4013

5017

5014

0018

0014

1018

1014

2018

2014

3018

3014

4018

4014

5018

5015

0019

0015

1019

1015

2019

2015

3019

3015

4019

4015

5019

50

Nam

e:

Dat

e:


APPENDIX 1








What controls are present in the experimental design and why?



In your groups 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 data



Outcome 3: Advice to candidates

Writing a report on an experiment in Biology

This advice is designed to help you write a report to meet theperformance criteria of Outcome 3. You must have played an active partin setting up the experiment and in collecting results.

When writing your experimental report avoid the use of the words ‘I’ or‘we’. That is instead of saying ‘we examined slides under themicroscope’ say ‘slides were examined under the microscope’. Alwaysuse the past tense in writing reports.

It is useful to structure your report under specific headings to avoidmissing out important sections. Write your report using the followingheadings and pay attention to the advice under each heading.

Title CheckUse the title in the Student Activity Guide.

AimA brief statement of the purpose of the experiment. ❒

MethodWrite a brief description of how the experiment was carried out. Do notput in too much detail, just sufficient so that anyone reading your reportwould know what you did rather than be able to repeat the experimentexactly.You should give the following information (as appropriate):

• labelled diagram or description of the apparatus, instruments used• variable altered• control measures used• measurements taken or observations made. ❒

ResultsRecord your raw data in a clear table with correct headings, appropriateunits and results/readings entered correctly. ❒

APPENDIX 2


APPENDIX 2

Analysis and presentation of resultsYou should analyse and present your results using one or more of thefollowing:

• a table with suitable headings and units, showing averages or otherappropriate computations

• a graph presented as a histogram, bar chart, connected points, line ofbest fit as appropriate, with suitable scales and axes labelled withquantity and units, and with data correctly plotted

• a scatter diagram or equivalent. ❒

ConclusionYour conclusion should use evidence from your experiment and relateback to the aim of the experiment. You should include at least one ofthe following:

• overall pattern to readings or observations (raw data)• trends in analysed information or results• connection between variables and/or controls. ❒

EvaluationThe evaluation could cover all stages of the activity including preparingfor the activity, analysis of the activity, and the results of the activity. Yourevaluation must include supporting argument in at least one of thefollowing:

• effectiveness of procedures• control of variables• limitations of equipment• possible sources of error• possible improvements.


APPENDIX 3

Outcome 3: Teacher/Lecturer Guide

All the performance criteria given in the left-hand column must beachieved in order to attain the outcome. The right-hand column givessuggestions which might aid the professional judgement of the assessor.

Performance criteria

a. The information iscollected by activeparticipation in theexperiment.

b. The experimentalprocedures aredescribed accurately.

c. Relevant measurementsand observations arerecorded in anappropriate format.

d. Recorded informationis analysed andpresented in anappropriate format.

Suggestions to aid professional judgement

The candidate has taken active part in thecollection of the information.

A clear statement of the aim of the experiment.A few brief concise sentences including asappropriate:• a labelled diagram or brief description of

apparatus, instruments used• how the independent variable was altered• control measures used• how measurements were taken or observations

made.There is no need for a detailed description. Theuse of the impersonal passive voice is to beencouraged as an example of good practice butthis is not mandatory for meeting the performancecriteria.

Readings or observations (raw data) must berecorded in a clear table with correct headings,appropriate units and results/readings enteredcorrectly.

Data should be analysed and presented in tabular,graphical format or as a scatter diagram orequivalent as appropriate:

• For a tabular presentation this may be anextension of the table used for performancecriteria c. above, and must include: suitableheadings and units showing averages or otherappropriate computations.

• For a graphical presentation this must include:data presented as a histogram, bar chart,connected points, line of best fit as appropriate,with suitable scales and axes labelled with quantityand units and with data correctly plotted.


APPENDIX 3

e. Conclusions drawn arevalid.

f. The experimentalprocedures areevaluated withsupporting argument.

Conclusions should use evidence from theexperiment and relate back to the aim of theexperiment. At least one of the following shouldbe included:• overall pattern to readings or observations (raw

data)• trends in analysed information or results• connection between variables and/or controls.

The evaluation could cover all stages of theactivity including preparing for the activity,analysis of the activity, and the results of theactivity. The evaluation must include supportingargument in at least one of the following:• effectiveness of procedures• control of variables• limitations of equipment• possible sources of error• possible improvements.