Biology
(Advanced Higher)
Practical Activities
7133
Summer 2000
HIGHER STILL
Biology
Advanced Higher
Practical Activities
Support Materials


CONTENTS
Introduction
Experimental work
 Purpose
 Structure
 Conditions required for Outcome 3
 Report writing
 Marking reports.
Practical activities
Cell and Molecular Biology
1.
2.
3.
4.
Staining a root tip and calculating its mitotic index
The effect of sucrose concentration on the growth of root explants of Sinapis alba
The effect of the end product, phosphate, on the enzyme phosphatase
Gel electrophoresis of DNA treated with restriction enzymes
Environmental Biology
5. Isolating and examining cysts of potato cyst nematodes (PCNs)
6. Examining mycorrhizae and the environmental factors affecting their abundance
7. The effect of relative humidity on the development of Botrytis infection in plants
Appendix 1 Preparing for the activity
Appendix 2 Outcome 3: Advice to Candidates
Appendix 3 Outcome 3: Teacher/lecturer Guide: Biology
ACKNOWLEDGMENT
These experiments were originally produced as a result of the Science and Plants for
Schools (SAPS) Biotechnology Scotland Project. Their assistance in the production of
these experiments is gratefully acknowledged.
Biology: Practical Activities (AH)
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Biology: Practical Activities (AH)
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INTRODUCTION
EXPERIMENTAL WORK
One report of an experimental activity is required as evidence for the assessment of
Outcome 3 in each unit. The choice of experiment is not prescribed in the unit
specification and so Centres can select from the activities included in the support
materials, adapt them for individual use, or use existing activities. The Student
Activity Guides provide guidance on the amount of detail and help students might
expect to receive. The experimental activity must allow for the collection and analysis
of information to meet the performance criteria of Outcome 3.
Outcome 3 performance criteria:
a. The information is collected by active participation in the experiment.
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.
e. 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 that could be developed
into investigations to meet the requirements of the Biology Investigation unit.
Any hazards associated with the experiments have been identified and suitable control
measures included in the support material as a result of risk assessment.
Structure
Teacher/lecturer guide
This includes a teacher/lecturer guide. This indicates whether the experimental
activity can be used to provide evidence for Outcome 3 or for the other purposes. A
section on background information includes the biology associated with the
experiment where necessary and any prior knowledge or skills students will require
before undertaking the activity. Advice on classroom management for the
teacher/lecturer will include advice on organising student groups, pooling results, time
required and the supply of materials to students. There will also be advice on possible
extension and follow up activities that could be developed into ideas for
investigations.
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Technical guide
This provides a list of materials required for each activity, including sources and
suppliers for items not generally available from major suppliers. There is advice on
the preparation of materials and risk assessments. The supply of materials to students
should allow for a degree of planning and organising of experimental work. This does
not mean planning and designing in the sense of an investigation as often the student
will be presented with an experimental procedure. Rather it should allow the student
to plan how he or she will lay out equipment and materials in preparation for carrying
out the experimental activity and planning the execution of the experimental
procedures.
Preparing for the activity
This section is designed to make students think actively about their experimental work
and to plan and organise its execution. To that end it includes an analysis of the
activity which poses questions about the experimental design. Students, although
presented with experimental procedures to follow are expected to plan and organise
carrying out the experimental work. In practical terms this will involve reading
through the procedure, identifying and collecting the materials they require and
organising themselves to carry out the procedures and record results either
individually or as a group. For some experimental activities ‘Preparing for the
activity’ has been customised by adding evaluation questions which will assist
students in considering issues which could be addressed in the experimental report.
This section presents a number of options for teachers and lecturers in teaching
experimental work. Students could be led through the stages in preparing for the
activity by their teacher/lecturer or it could be presented to students as an individual
or group activity. Alternatively the different stages in preparing for the activity could
be presented as a mixture of these approaches as teachers and lecturers consider
appropriate for their students. Also different experimental activities may lend
themselves to different approaches, or as students’ skills develop 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 are customised questions on evaluation
in which case a ‘Preparing for the activity’ section appears in the support material for
that particular activity.
Student activity guide
This includes an introduction, which provides background information for the student
on the biology of the activity or any other information required. The experimental
procedures for students are described in the equipment and materials section and the
instructions. The instructions take the students through the steps required for the
activity as well as limited advice on the recording, analysis and presentation of data.
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Conditions required for practical work for Outcome 3
Arrangements documentation and Subject Guides refer to assessment being carried
out under controlled conditions to ensure reliability and credibility. For the purposes
of internal assessment, this means that assessment evidence should be compiled under
supervision to ensure that it is the students’ own work.
It must be emphasised that the assessment for this outcome is not a special assessment
event but part of the on going learning and teaching process. The experimental
activity is likely to be performed by a small group of students together. After
collection of the experimental information each student must complete a report
individually under supervision. A written report should be provided for evidence
where circumstances make that possible. For students with special needs for whom
written evidence is not appropriate alternative forms of report can 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 is appropriate to support students
in producing a report to meet the performance criteria. Thus redrafting of reports after
necessary supportive criticism is to be encouraged as part of the learning and teaching
process and to produce the evidence for assessment. Redrafting should focus on the
performance criteria concerned and, as a general rule should be offered on a
maximum of two occasions following 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 the report to aid clarity and ensure
completeness of their report. This gives advice on structuring the report under
specific headings making a blank report booklet unnecessary. In some experiments
where only one of the items listed in the conclusion or evaluation is likely to be
required this can be indicated to the students.
Marking reports
The ‘Outcome 3 - Teacher/Lecturer Guide: Biology’ is included as Appendix 3. This
page summarises the performance criteria together with suggested items which might
aid the professional judgement of the assessor. It is important to consider each
individual experiment and how the specific advice given in the Teacher/lecturer guide
for the experimental activity relates to the suggestions to aid professional judgement.
Centres may wish to produce customised departmental marking schemes for the
particular practical activities they use to provide evidence of Outcome 3. The advice
on marking reports for Outcome 3 at Higher and Int 2 contained in the support
material Marking Advice for Assessing Outcome 3 (Int 2 and H) 5722 published Aug
1999 applies equally to Advanced Higher Biology.
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The final decision on achievement must be on the basis of the performance criteria.
Although poor grammar, poor sentence construction and bad spelling would be drawn
to the student’s attention, these aspects are not in any of the performance criteria.
Definitive guidance on the assessment of student reports from Outcome 3 is to be
found in National Assessment Bank materials.
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Unit: Molecular Biology (AH): Plant Tissue Culture
Title: The effect of sucrose concentration on the growth of root explants of Sinapis
alba
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 tissue culture methods
 develop practical skills in aseptic techniques used in tissue culture
 develop problem solving skills and in particular Outcome 2 PCs:
(c) conclusions drawn are valid and explanations given are supported by evidence.
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
Tissue culture is the basis of a multi-million pound industry. This industry can
provide almost infinite numbers of genetically identical, disease-free plants which can
subsequently be used in research, agriculture or sold to the general public.
In the past, school tissue culture methods have relied on callus formation using carrot
or cauliflower. However, contamination was a common problem. The technique
described here uses explants grown from surface sterilised seed, greatly reducing the
chances of systemic infection. This method also avoids a lengthy incubation period.
Thus, even if infections are present, it should be possible to obtain results before they
are obscured by growth of the interfering micro-organism. The experimental results
will hopefully lead to discussions on genes being ‘switched on’ and the idea of
totipotency (each cell contains all the genes of an organism).
Some 35 mm slides and accompanying notes will be made available to show
examples of experimental results and also the commercial importance of tissue culture
techniques.
Classroom management
Students can work individually or in pairs for this experiment.
Sterilising and sowing the seed will take about 30 minutes. The seeds then require 45 days, preferably under a lightbank, to be suitable as a source of explants. Taking
explants and transferring them to various sterile media will require a further 50-60
minutes. The plates then need to be examined daily for about 7-10 days.
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Supply of materials
In order to satisfy the core skill in problem solving, students will be required 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 PCs b-f.
PC b: to include sterilisation of seeds: preparing and transferring explants to
prepared agar plates; precautions taken to prevent contamination
PC c: a table of results with appropriate headings and units showing length of root
tips and number of lateral roots formed in each mid root explant.
PC d: a graph of length of the root tips after a measured length of time and % sucrose in the
media is drawn and/or a graph of number of lateral roots formed in mid-root
explants and % sucrose in the media is drawn.
PC e: a conclusion is made which fits the graph drawn e.g. the higher the % sucrose
present the greater the increase in length of the explant.
PC f: evaluation points include:
 the effectiveness of the aseptic techniques used
 difficulties encountered measuring the length of the root tips
 the number of explants used to obtain an average and thus form a conclusion.
 evidence of explant cells being totipotent.
Extension work
There are several easily controlled variables in this experiment making it ideal for
project work.
(i)
The type of explants - root tips, mid-root and mid-hypocotyl explants are all
available.
(ii)
The medium that the explants are in contact with:
- vary the concentration of MS salts (e.g. 0, 1.1 g/L, 2.2 g/L, 3.3 g/L)
- vary the concentration of sucrose (e.g. 0, 1% - 5%)
- vary abiotic factors e.g. temperature, light intensity
- introduce plant growth substances e.g. auxins or kinetins (see Technical
Guide)
- introduce silver nitrate (100 mg/L) which is thought to inhibit ethylene
production.
Other explant features:
- does regenerative ability vary with age of explant?
- do mid-root explants near the root tip have greater regenerative powers than
those further from the root tip?
(iii)
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References
Hanley-Browne, M. (1998) Fast tissue culture. Biological Sciences Review, 10(3), 26.
Fuller, M. P. and Fuller F. M. (1995) Plant tissue culture using Brassica seedlings.
Journal of Biological Education, 29(1), 53-59.
Acknowlegements
This practical was based on work initially carried out by M. Fuller, University of
Plymouth, F. Fuller, South Devon College and M. Hanley-Brown, Charterhouse
School.
Web sites
The National Health Museum
http://www.accessexcellence.org
Kitchen Culture Kits
http://www.home.turbonet.com/kitchenculture
Plant Tissue Culture Information Exchange
http://aggiehorticulture.tamu.edu/tisscult/tcintro.html
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
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Unit: Molecular Biology (AH): Plant Tissue Culture
Title: The effect of sucrose concentration on the growth of root explants of Sinapis
alba
TECHNICAL GUIDE
Materials required
Materials required by each student/group:
20 white mustard seeds
beaker
20 cm3 10% bleach
sterile water
70% alcohol for sterilising instruments
forceps
scalpel
sterile water agar in a jar with lid
4 sterile petri dishes
petri dish containing sterile agar with MS salts
petri dish containing sterile agar with MS salts and 0.5% sucrose
petri dish containing sterile agar with MS salts and 1.0% sucrose
petri dish containing sterile agar with MS salts and 3.0% sucrose
bunsen
ceramic mat
marker pen
piece of graph paper
piece of black paper
tape
hand lens
Preparation of materials
10% bleach - use unthickened bleach
Jars to grow seedlings - 24 pack of vessels for plant tissue culture available from
Sigma -Catalogue Number V8630, £9.90. Baby food jars or small jam jars would also
be suitable but the lid must be easily removed and replaced and make a good seal.
Prior to adding water agar, sterilise jars by autoclaving at 120C for 15 minutes.
Water agar - Each jar requires about 40 cm3. Each group will require at least one jar.
Add 1.4 g of agar (see table below) per 200 cm3 water. Stir and then autoclave at
120C for 15 minutes. This is enough for five jars. Each group of students will
require a jar.
Chemicals for making up media for explants are available from Sigma, Fancy Road,
Poole, Dorset BH12 4QH. Tel: 0800 717181 Fax: 0800 378538
Biology: Practical Activities (AH)
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Product
Catalogue
Quantity
Cost
number
(1999)
*Agar
A-1296
100g
£18.30
MS mineral salts
M-5524
1 sachet to make 1 L £1.30
** indole-3-butyric acid (IBA) I-5386
1g
£7.50
** kinetin solution (1 mg/ml)
K-3253
100 cm3
£10.50
* this agar is specific for plant tissue culture experiments
** only required for project work - not for the basic experiment.
Mustard seeds are available from Philip Harris, garden centres or health food shops.
Making up the medium for explants
The instructions below are enough to make five plates of four different media enough for 5 groups of students.
Dissolve 0.88g of Murashige & Skoog (MS) mineral salts in 400 cm3 of distilled
water. Stir and test for pH. Adjust the pH to between 5.6 and 6.0 using a few drops of
dilute NaOH or HCl. Divide into four batches of about 100 cm3.
Add 0.8 g agar to batch one (control).
Add 0.8 g agar and 0.5 g sucrose to batch two.
Add 0.8 g agar and 1.0 g sucrose to batch three.
Add 0.8 g agar and 3.0 g sucrose to batch four.
Label each batch appropriately. Autoclave for 15 minutes at 121C and 15 psi.
Label five sterile petri dishes ‘CONTROL - 0% SUCROSE’ and once cooled sufficiently
pour about 20 cm3 of the control liquid into each dish. Allow to cool and solidify.
Label five sterile petri dishes appropriately each time and repeat this procedure with
the other sterile liquids. Once set, store the petri dishes upside down in a refrigerator
until required.
For project work
This medium will allow callus formation and hypocotyls (stem explants) may produce
new shoots and form roots.
Basic medium: 8 g/l agar, 4.4 g/L MS salts, 30 g/L sucrose and adjust pH to between
5.6 and 6.0 using dilute HCl or NaOH.
Make up in four equal batches and add:
Batch 1 - nothing
Batch 2 - 2 mg/L IBA (auxin)
Batch 3 - 4 mg/L Kinetin (cytokinin)
Batch 4 - 2 mg/L IBA and 4 mg/L Kinetin
Autoclave for 15 minutes at 121C and 15 psi, allow to cool to about 60C and decant
into labelled sterile petri dishes.
NOTE: Dissolve the IBA in a drop of dilute HCl initially, then add the appropriate
volume of water. Plant growth substances stock solutions can be stored in the
refrigerator for up to two months.
Wear gloves when handling the plant growth substances.
Biology: Practical Activities (AH)
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Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
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Unit: Molecular Biology (AH): Plant Tissue Culture
Title: The effect of sucrose concentration on the growth of root explants of Sinapis
alba
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity
 What is the aim of the activity?
 What is being varied in the activity?
 What variables must be kept constant?
 What measurements are you going to make?
 What controls are being used and why?
Getting organised for experimental work
For success in this experiment, the explants and media they grow on must be sterile.
Are you aware of the techniques used to:
(i) produce sterile seedlings
(ii) produce sterile media for the explants to grow on
(iii) prevent air-borne micro-organisms from contaminating the petri dishes while
carrying out the experiment?
In your group decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active 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
Were the precautions to avoid air-borne contamination sufficient?
Were there sufficient numbers of explants for a meaningful conclusion to be formed
Could the increase in length of the root tips be measured with sufficient accuracy?
Do the results suggest that genes have been switched on and that cells are totipotent
i.e. each cell carries all the genes necessary to build the entire organism?
Biology: Practical Activities (AH)
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Unit: Molecular Biology (AH): Plant Tissue Culture
Title: The effect of sucrose concentration on the growth of root explants of Sinapis
alba
STUDENT ACTIVITY GUIDE
Introduction
Plant tissue culture can be carried out in many different ways. All the different
methods use explants (i.e. small pieces of sterile plant material) grown in a sterile
medium. It is used widely in research and industry for some of the following reasons:
 To eliminate viruses from infected plants and thus increase vigour e.g. potatoes,
strawberries
 To achieve large numbers of genetically identical plants when conventional
propagation is too slow or difficult
 To produce new varieties of plants e.g. mutations occurring during mitosis can be
expressed or protoplast fusion allowed to occur
 To produce haploid plants useful for genetic studies
In this method you are going to surface sterilise seeds of white mustard (Sinapis alba)
and allow them to grow in sterile water agar for several days. Explants will then be
removed from the seedlings and transferred to media with various amounts of sucrose
in it. The explants will then be examined regularly for signs of growth.
Some 35 mm slides may be available to show possible experimental results and
examples of tissue culture carried out commercially.
Equipment and materials
Materials required by each student/group:
20 white mustard seeds
beaker
20 cm3 10% bleach
sterile water
70% alcohol for sterilising instruments
forceps
scalpel
sterile water agar in a jar with lid
4 sterile petri dishes
petri dish containing sterile agar with MS salts
petri dish containing sterile agar with MS salts and 0.5% sucrose
petri dish containing sterile agar with MS salts and 1.0% sucrose
petri dish containing sterile agar with MS salts and 3.0% sucrose
bunsen
ceramic mat
marker pen
Biology: Practical Activities (AH)
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piece of graph paper
piece of black paper
tape
hand lens
Instructions
1.
Put 20 white mustard seeds in a beaker and cover with 10% bleach for 10-15
minutes.
Wear eye protection when handling bleach; spilt bleach
will affect clothing.
2.
Pour off bleach and rinse seeds three times with
sterile water.
3.
Dip forceps in 70% alcohol and hold in a blue
bunsen flame for about 5 seconds to sterilise them.
Allow to cool and use the forceps to transfer the
surface-sterilised seeds onto the surface of water
agar in a sterile jar. Hold the lid over the jar to
reduce accidental contamination from air-borne
micro-organisms. Resterilise the forceps after
sowing every fifth seed.
4.
Secure the lid and place under a lightbank or on a
sunny windowsill for 4-5 days.
After 4-5 days
Aseptic techniques are used in this experiment. The following precautions must
therefore be followed.
i) When moving to a new seedling always sterilise the forceps and the scalpel. If
you suspect an instrument is still hot when required plunge it into the water agar
in the tub of seedlings for a few seconds.
ii) Open any container a minimum distance for a minimum length of time. The lid
then acts as a ‘shield’ and reduces the chances of air-borne micro-organisms
contaminating your plates.
iii) Take a new empty sterile petri dish for cutting the seedlings after you have ‘filled
up’ each petri dish with 4 mid-roots and 4 root tips.
iv) If the sterile part of your instruments accidentally touch something not sterile e.g.
bench, clothing, immediately resterilise the instrument. Discard any seedlings
which accidentally touch non-sterile objects.
v) Use a room with no draughts. All windows and doors should remain closed.
5.
Sterilise forceps as in Step 3 and rest them on e.g. a pencil, so that the tips are
not touching the bench. Meanwhile, sterilise a sharp scalpel in the same way.
Biology: Practical Activities (AH)
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6.
Lift the lid of the jar containing the
seedlings and use the sterile forceps to
transfer a seedling to an empty sterile
petri dish sitting on a piece of graph
paper. Replace both the lid of the jar
and the petri dish immediately.
7.
With the lid of the petri dish acting as a
shield, use the sterile scalpel to cut off
the last 3 mm of the root. Obtain the
mid-root explant by cutting off the next
5 mm of root , as shown in the
diagram.
8.
Use sterile forceps to transfer the two sections of root to a petri dish containing
agar and MS salts. The sections of root will show up more clearly if this petri
dish is sitting on a black background.
9.
Repeat steps 6-8 until the petri dish has four
root tips and four mid-root explants, as shown
in the diagram. Remember to sterilise the
forceps and scalpel before starting on each new
seedling.
10.
Collect petri dishes containing:
1. agar + MS salts + 0.5% sucrose
2. agar + MS salts + 1.0% sucrose
3. agar + MS salts + 3.0% sucrose
11.
Transfer four root tips (3 mm in length) and
four mid-roots (5 mm in length) to each of these
plates. Remember to follow the precautions
required to do these procedures aseptically.
12.
Seal all dishes with tape. With a marker pen, put a small dot on the base of the
dish at either end of each root tip. This will make any increase in length easier
to measure. Dishes must NOT be re-opened. With a marker pen, label each
petri dish with the date.
13.
Store the petri dishes, with lids uppermost, in a warm, dark cupboard or in an
incubator at 25C.
14.
The next day, examine the root tips and record any increases in length. To do
this, place a transparent ruler on a white sheet of paper. Place plate containing
root tips above millimetre scale on ruler. Use a hand lens if necessary.
Continue to do this daily, if possible, for the next week.
15.
With the mid-roots, record the number of lateral roots formed on each explant
after 7-10 days.
Biology: Practical Activities (AH)
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16.
Record your results in a table with appropriate headings and units.
17.
Average the increase in length of root tip for each petri dish. Take a total count
of number of lateral roots on the mid-root explants for each dish.
18.
Present your results as a graph with suitable scales and axes labelled with
quantities and units.
Note 1: When plant growth substances are added to the growth medium regeneration
of complete plants can be achieved after a few weeks, thus demonstrating
totipotency. Such plants are initially vulnerable as:
(i) being provided with sugar makes their photosynthetic ability poor.
(ii) being grown in 100% humidity, no waxy cuticle is present nor do stomata
close in response to low humidity.
The plants must therefore be carefully acclimatised in controlled fogging
chambers.
Note 2: In industry, to maintain sterile conditions when working with explants, air is
filtered to remove all micro-organisms and gently passed over the working
area to remove any contaminants. The room is also under positive air
pressure so that if a door is opened no air from the adjoining room will enter.
Operators wear lab coats, which are sterilised regularly, and hairnets to
prevent micro-organisms from hair dropping onto the sterile plant material.
Biology: Practical Activities (AH)
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Unit: Cell and Molecular Biology (AH): Structure, function and growth 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 PCs:
(b) information is accurately processed, using calculations where appropriate
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
In this activity students will prepare and stain root tips. To provide evidence for
Outcome 3 students must either have TWO different sources of root tips OR stain one
type of root tip with two different stains. A comparison between EITHER the root
types OR the stains will then be possible.
Two recommended sources of roots are garlic and hyacinth. The garlic cloves, bought
normally for cooking purposes, will produce roots at any time of year. Hyacinth
bulbs can be bought at Garden Centres during autumn and winter. Both garlic cloves
and hyacinth bulbs will produce ample roots for the experiment.
Suitable stains for studying the stages of mitosis in root tips are lactopropionic orcein
and toluidene blue.
The mitotic index is the fraction of cells in a microscope field which contain
condensed chromosomes. This index will be calculated for each slide prepared.
Preparation of the plant materials and the stains is covered in the Technical Guide.
To make this activity non-seasonal, it is possible to ‘fix’ the root tips when available
and then store them until required. Fixing of root tips is only covered in the Technical
Guide.
Classroom management
Students are asked to mark the root tip one or two days prior to staining the root tips.
This will enable them to link rate of growth with mitotic index.
Microscopic examination of the slides:
Students should examine several slides and calculate the mitotic index for each one.
Prepared slides could also be available.
Biology: Practical Activities (AH)
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Supply of materials
In order to satisfy the core skill in problem solving, students will be required 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 PCs b-f.
PC 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 the different stages of
mitosis; the magnification used should also be noted
PC d: a table of results recording:
(i) the number of cells containing condensed chromosomes in 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 microscope fields for each
situation (i.e. two for each type of root tip or two for each stain used)
PC e: Either a conclusion is made about the rate of mitosis in the different types of
root tips (the higher the mitotic index the greater the rate of mitosis) OR a
conclusion is made about the efficiency 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 too short a time, maceration
will be difficult; if too long a time the tip 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 orcein usually gives better
definition of chromosomes while the toluidine blue is stronger in colour
 the condition of the roots and their rate of growth prior to using them for the
experiment
Extension work
Try to vary the mitotic index of the plant tissue e.g. cutting the root tips and keeping
them at 0C for 24 hours may increase the mitotic index. The experimental method
can be varied e.g. varying the temperature or concentration of acid; varying the time
the root tip is in the acid; squashing the root tip with a coverslip instead of
macerating; varying the age of the root used; preparing the stains differently (e.g.
different dilutions, different pHs); heating the lactopropionic orcein slide gently;
investigating a possible link between rate of growth of root and mitotic index.
Biology: Practical Activities (AH)
19
Acknowledgements
Information and advice from Dr Kwiton Jong, Royal Botanic Garden, Edinburgh, is
gratefully acknowledged. Information was also received from Ashby Merson-Davies,
Sevenoaks School, Kent.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
Biology: Practical Activities (AH)
20
Unit: Cell and Molecular Biology (AH): Structure, function and growth of
prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
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 protection
compound microscope (x100 - x400 magnification)
small beaker of 1M hydrochloric acid (2 will be required if plant material is being
investigated)
small beaker of water and dropper
microscope slides
coverslips
fine forceps
dissecting needle
scissors
soft tissue paper
ruler
fine thread
dropping bottle of lactopropionic orcein AND/OR (see below) dropping bottle of
toluidine blue
garlic clove with suitable roots AND/OR (see below) hyacinth bulb with suitable
roots
Materials to be shared:
water bath at 60C
marker pen
timer
dropping bottle of 50% glycerol
dropping bottle of 70% ethanol
lens tissue
Preparation of materials
If PLANT MATERIAL is to be varied prepare BOTH plant types below. If STAIN is
to be varied prepare just one of the plant types.
Biology: Practical Activities (AH)
21
To prepare hyacinth bulb roots: Place the bulb in a suitably sized container with
water so that the root end is just in contact with the water. It is best to change the
water daily if possible. Roots of a suitable length (2-6 cm) will be available within a
week and perhaps sooner.
Hyacinth bulbs can cause allergies. Wear gloves if handling the bulbs regularly.
To prepare garlic clove roots: Carefully peel the clove and place it in a suitably sized
container with water e.g. test tube/boiling tube so that the root end is just immersed in
the water. It is best to change the water every 2-3 days. Roots of a suitable length (26 cm) will be available after 2-4 days).
If STAIN is to be varied prepare BOTH stains, as detailed below. If PLANT
MATERIAL is to be varied prepare 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 distilled water.
Toluidine blue is harmful if swallowed. Prepare a 0.5% solution in a
citrate/phosphate buffer at pH4 (20 cm3 0.1M citric acid + 10 cm3 disodium hydrogen
phosphate + 8 cm3 distilled water).
Fixing the roots
This stage is required only if suitable roots are available but they are to be stained at a
later date.
Mix 6 cm3 absolute alcohol with 2 cm3 glacial acetic acid in a fume cupboard. This
mixture is called Farmer’s fluid and must be freshly prepared. Once added to the
Farmer’s fluid, the root tips can be stored for many months in a refrigerator.
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
Biology: Practical Activities (AH)
22
Unit: Cell and Molecular Biology (AH): Structure, function and growth of
prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
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?
Decide what a ‘nucleus’ should look like for it to be composed of condensed
chromosomes.
In your group, decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active 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 and appropriate
headings.
Evaluation
If varying plant material, was rate of growth of the two roots similar? If not, 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 to absorb the
stains? Were they absorbed too much/insufficiently?
Does the mitotic index vary much between different results? Account for these
differences, if possible.
Was the treatment in acid (step 4) sufficient to allow for both easy handling of the
root tip and easy maceration?
Biology: Practical Activities (AH)
23
Unit: Cell and Molecular Biology (AH): Structure, function and growth of
prokaryotic and eukaryotic cells
Title: Staining a root tip and calculating its mitotic index
STUDENT ACTIVITY GUIDE
Introduction
You are going to stain root tips and examine them for signs of cells dividing by
mitosis. The chromosomes inside the nuclei of such cells condense and become
visible. You should know what condensed chromosomes look like and how they
move about inside a cell when undergoing mitosis.
Equipment and materials
Materials required by each student/group:
gloves and eye protection
compound microscope ( x100 - x400 magnification)
small beaker of 1M hydrochloric acid (2 will be required if plant material is being
investigated)
small beaker of water and dropper
microscope slides
coverslips
fine forceps
dissecting needle
scissors
soft tissue paper
ruler
fine thread
dropping bottle of lactopropionic orcein AND/OR (see below) dropping bottle of
toluidine blue
garlic clove with suitable roots AND/OR (see below) hyacinth bulb with suitable
roots
Materials to be shared:
water bath at 60C
marker pen
timer
dropping bottle of 50% glycerol
dropping bottle of 70% ethanol
lens tissue
Biology: Practical Activities (AH)
24
Wear gloves and eye protection whilst carrying out this experiment. Avoid skin
contact with the stain(s) and avoid breathing in the fumes of the stain, lactopropionic
orcein, if used.
Instructions
Either two types of roots OR two different stains will have been prepared. Find out
what is available.
1.
One or two days before staining the root tips, remove the plant material
carefully from the water and blot dry gently. Use a permanent marker pen to
mark a small dot about 2mm from the end of some root tips. Replace the plant
carefully in the water.
2.
After one to two days, remove the plant material and use the thread and ruler
to measure how much the root tips have grown since marked.
3.
Preheat about 10 cm3 of IM hydrochloric acid in a small beaker to 60C using
a waterbath. Meanwhile, use a lens tissue and alcohol to clean microscope
slides and coverslips.
4.
Using scissors remove the last 2mm from several young vigorously growing
root tips. Place them in the preheated acid and return to the waterbath for 4-5
minutes.
5.
Gently transfer each root tip to a clean microscope slide containing a large
drop of water.
6.
Gently blot dry with a piece of soft tissue.
7.
Using a dissection needle, thoroughly macerate the root tip and spread 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 tip and
IMMEDIATELY cover with a coverslip, invert the slide and blot firmly
several times on a wad of tissues.
If using lactopropionic orcein - Add one drop to the macerated root tip and
leave for 3-4 minutes. To speed up absorption of the stain, warm the slide
gently by holding it 30-40cm above a yellow bunsen flame (if your hand
becomes uncomfortable you are heating the slide too much). Cover with a
coverslip, invert the slide and blot firmly several times on a wad of tissues.
9.
View under a microscope, x40 - x100 magnification initially. Scan the slide to
locate the region of mitosis.
Biology: Practical Activities (AH)
25
10.
View this area at a higher magnification (x400 should be sufficient) and count:
(i) the total number of cells in the microscope field
(ii) the number of cells with condensed chromosomes which are going through
any of the four stages of mitosis. You will have to decide where your cut-off
point is when considering if cells in prophase and telophase contain condensed
chromosomes (consult textbooks).
11.
Repeat steps 9 and 10 for the various microscope slides prepared. If you want
to prevent the slides from drying out, mount them in 50% glycerol.
12.
Calculate the mitotic index for each slide examined (the mitotic index is the
fraction or percentage of cells containing condensed chromosomes).
13.
Draw a table with suitable headings summarising your results.
14.
Compare your results with other groups.
Biology: Practical Activities (AH)
26
Unit: Cell and Molecular Biology (AH): Molecular interactions in cell events:
Catalysis
Title: The effect of the end product, phosphate, on the enzyme phosphatase
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 effect of a product on the activity of
the enzyme that produced it
 develop problem solving skills and in particular Outcome 2 PCs:
(c) conclusions drawn are valid and explanations given are supported by evidence
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
Phosphatases are a group of enzymes which release phosphate groups for cell
metabolism. The resulting phosphates can then be used for a variety of purposes e.g.
incorporated into the nucleotides of DNA and RNA, to make phospholipids - a
component of cell membranes or to make energy rich ATP.
phosphatase
various substrates
phosphates
nucleic acids
phospholipids
ATP
Phosphatases are thus key enzymes in cell metabolism.
There are two main groups of phosphatases, acid or alkaline depending on their
optimum pH. This experiment involves an acid phosphatase extracted from
germinating mung beans (beansprouts). The enzyme is also found in potatoes, tomato
leaves, wheatgerm and in the seeds of many legumes.
A simple aqueous extract, derived from beansprouts is used as the enzyme solution.
An artificial substrate, phenolphthalein phosphate is used. It is colourless in acid pH.
Various concentrations of a phosphate salt are added to a series of test tubes
containing the enzyme and the substrate. The phosphate salt, being an end-product,
also inhibits the enzyme. This experiment will demonstrate that the higher the
concentration of phosphate present the greater the inhibition of the enzyme.
phosphatase
phenolphthalein phosphate
phenolphthalein
+ phosphate
end-product inhibition
Biology: Practical Activities (AH)
27
Any free phenolphthalein released can be estimated by the addition of alkali. The
alkali does two things:
a) it stops the reaction by denaturing the phosphatase
b) the alkaline pH turns the free phenolphthalein pink.
The intensity of the pink colour produced can be measured quantitatively using a
colorimeter.
Classroom management
Students can work individually or in pairs for this experiment.
The Student Activity Guide asks students to calculate the mass of sodium phosphate
to add to achieve certain molarities. Although not required for purposes of
assessment it was thought to be a useful and not too difficult task for students to
undertake. However, if the class teacher so desires, the students or technicians can be
given the masses, bypassing this step in the experiment.
beaker number
molarity of sodium phosphate
(NaH2PO4) added to buffer
1
2
3
4
5
0
0.05M
0.10M
0.20M
0.30M
mass of sodium phosphate
(NaH2PO4) added to buffer
/100 cm3
0
0.69 g
1.38 g
2.76 g
4.14 g
Estimated times: about one hour including a 20 minute incubation period.
There is no point at which it is suitable to leave the experiment overnight. However,
any colour formed on addition of the sodium carbonate solution is stable. It is
therefore possible to store the coloured tubes in a refrigerator overnight and read the
% transmission/absorbance the following day.
If the school does not have a suitable centrifuge this step can be missed out. Instead,
filter the beansprout extract through a double layer of muslin. If the tubes are pale
pink, colorimeter readings may be unreliable. Students could refer to this discrepancy
in the evaluation part of their report.
In plotting class results the concept of error bars can be introduced (see instruction 16)
Supply of materials
In order to satisfy the core skill in problem solving, students will be required to
identify and obtain resources required for themselves. Further advice on supply of
material is given in the Technical Guide.
Biology: Practical Activities (AH)
28
Advice on marking Outcome 3 report
Specific advice for PCs b-f.
PC b: to include the preparation of the enzyme; the contents of each test tube; the
factor being varied (phosphate concentration); other conditions kept constant (e.g. pH,
temperature, incubation time, age of plant material); the two roles of the sodium
carbonate.
PC c: a table of results with appropriate headings (molarity of sodium phosphate and
% transmission)
PC d: results are graphed with molarity of sodium phosphate on the x-axis and %
transmission on the y-axis; an appropriate scale is used and axes are labelled; the
points are correctly plotted and a line of best fit is drawn.
PC e: a conclusion is made which fits the graph drawn e.g. as phosphate
concentration increases the activity of the enzyme decreases showing end product
inhibition.
PC f: evaluation points include:
 the need for replicates to determine the reliability of the result in each test tube
 the slight variation in pH between each test tube due to the addition of different
molarities of sodium phosphate
 how possible variables have been controlled and why it is important to control
these factors
 the need to prevent cross contamination between phosphate solutions and how it is
avoided
 accounting for the differences in the results between groups
Extension work
 Compare phosphatase activity
 in plant material as it matures
 in different parts of the plant material
 in different plants.



Investigate the properties of the enzyme by varying temperature, pH and substrate
concentration.
Investigate the rate of reaction by varying the times at which the reaction is
stopped.
Investigate the inhibiting effect of other phosphates or of a general enzyme
inhibitor such as a lead salt.
Biology: Practical Activities (AH)
29
References
Meatyard, B.(1999) Phosphatase enzymes from plants., Journal of Biological
Education, 33(2), 109-112.
Larkcom, J. (1991) Oriental Vegetables 60-61. John Murray ISBN 0-7195-4781-4
This practical was based on work initially carried out by Dr Barry Meatyard, SAPS,
Warwick Institute of Education, Warwick University, Coventry CV4 7AL.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
Biology: Practical Activities (AH)
30
Unit: Cell and Molecular Biology (AH): Molecular interactions in cell events:
Catalysis
Title: The effect of the end product, phosphate, on the enzyme phosphatase
TECHNICAL GUIDE
Materials required
Materials required by each student/group:
mortar and pestle
filter funnel
piece of muslin (approx. 12 cm x 12 cm)
centrifuge tube
test tube
5 boiling tubes with rack
marker pen
2 x 5 cm3 or 10 cm3 syringe
2 x 1 cm3 syringes or pipettes
stirring rod
at least 5 cm3 1% phenolphthalein phosphate (PPP) solution
beaker containing at least 25 cm3 10% sodium carbonate solution
gloves and eye protection
Materials to be shared:
water bath at 30C
at least 500 cm3 buffer solution pH 5
5 beakers each with an accurate 100 cm3 graduation mark
spatula
balance
weighing boats/filter paper
Sodium phosphate (NaH2PO4)
5 droppers
500g packet of beansprouts
bench centrifuge
colorimeter with 550nm filter and cuvettes or test tubes as appropriate.
Preparation of materials
Wear gloves when preparing the PPP solution and the buffer.
Phenolphthalein phosphate (PPP) can be obtained from Sigma - catalogue no. P 9875
- 1999 price - £6.90 for 1g. Care should be taken when weighing as the dust may be
hazardous. The 1% solution should be made up just before use or if necessary the day
before and stored overnight in the refrigerator. PPP slowly degrades to free PP in
solution.
Biology: Practical Activities (AH)
31
N.B. TWO different sodium phosphates are used in this experiment. The dibasic form
(Na2HPO4) is used as a component of the background buffer which is added to all
tubes. The monobasic form (NaH2PO4) is added in varying amounts to the
background buffer. The monobasic form is used to inhibit the enzyme.
Sodium phosphate (NaH2PO4)(the enzyme inhibitor) can be obtained from Sigma catalogue no. S 9638 - 1999 prices - £9.40 for 250g. Although other phosphates
e.g.Na3PO4, Na2HPO4, K2HPO4 will also inhibit phosphatase they will tend to change
the pH considerably when added to the buffer. Such changes in pH must be rectified
by adding a few drops of 5M hydrochloric acid while checking the solution with a pH
meter. Using NaH2PO4 as the inhibitor avoids readjusting the pH as even at the
highest concentration used (0.3M) the pH will vary as little as 0.1 - 0.2.
To make up 100 cm3 of buffer add 51.5 cm3 0.2M Na2HPO4 (the dibasic sodium
phosphate) to 48.5 cm3 0.1M citric acid. The pH of this mixture should be close to
5.0. Adjust to exactly 5.0 by adding the appropriate solution drop by drop while
checking with a pH meter.
Teachers may wish to get technicians rather than students to add the monobasic
sodium phosphate (NaH2PO4) to the buffer. If this is the case follow the instructions
below:
1. Label 5 beakers 1 - 5.
2. The molarities of sodium phosphate (NaH2PO4) to be present in each beaker is
shown in the table. Also, shown is the mass of sodium phosphate required in 100
cm3 of buffer to achieve this molarity.
beaker number
molarity of sodium phosphate
(NaH2PO4) added to buffer
1
2
3
4
5
0
0.05M
0.10M
0.20M
0.30M
mass of sodium phosphate
(NaH2PO4) added to buffer
/100 cm3
0
0.69 g
1.38 g
2.76 g
4.14 g
Weigh out the appropriate mass of sodium phosphate for each molarity. Add it to
the beaker along with about 90 cm3 buffer solution.
3. Stir until it is completely dissolved and then with a dropper carefully add more
buffer until the solution reaches the 100 cm3 mark.
It will usually be more convenient to buy a 500g packet of beansprouts from a
supermarket. The Student Activity Guide has been written assuming that such
beansprouts are being used as the source of phosphatase.
Biology: Practical Activities (AH)
32
Alternatively, soak mung bean seeds in a shallow dish and pour off excess water.
Rinse two times each day with fresh water and drain. Too much water will cause
them to rot, too little and they will dry out. Leave in a dark cupboard at about 25C
and harvest after 3 days. When making the enzyme solution from this source 1 cm3 of
water should be added for every seedling used. Most of the water should be added
after the seedlings have been crushed using a mortar and pestle.
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
Biology: Practical Activities (AH)
33
Unit: Cell and Molecular Biology (AH): Molecular interactions in cell events:
Catalysis
Title: The effect of the end product, phosphate, on the enzyme phosphatase
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity





What is the aim of the activity?
What is being varied in the activity?
What variables must be kept constant?
What measurements are you going to make?
What controls are being used and why?
Getting organised for experimental work
What safety measures are you required to take?
In your group decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active part in setting up the
experiment and in collecting results.
You may be asked to calculate the mass of sodium phosphate required to obtain a
solution with a certain molarity. If you do not know how to work this out consult the
Supplementary Student Information sheet.
Recording of data
Prepare tables to record your group results.
You should use a ruler, correct headings and appropriate units.
Evaluation
The pH of the test tubes you will set up will vary slightly (0.1 - 0.2) due to the
different concentrations of sodium phosphate. How could you demonstrate how
important this variation in pH is? What could you do to make the pH of all the test
tubes exactly the same?
What experimental conditions should be kept constant and why?
How much variation is there between the results of different groups? If this is
considerable how could you take/account of this before plotting the graph?
Biology: Practical Activities (AH)
34
Unit: Cell and Molecular Biology (AH): Molecular interactions in cell events:
Catalysis
Title: The effect of the end product, phosphate, on the enzyme phosphatase
STUDENT ACTIVITY GUIDE
Introduction
Phosphatase enzymes release phosphates from a variety of substrates. These
phosphates are required for synthesis of, for example, ATP, phospholipids and
nucleotides. They are found in both plant and animal tissues and can be classified as
acid or alkaline depending on their optimum pH. This experiment uses an acid
phosphatase (optimum pH 5) from germinating mung beans (beansprouts). The same
type of enzyme can be found in many other plants including potatoes, legume seeds
and tomato leaves.
In this experiment the enzyme is obtained by grinding the beansprouts and collecting
the liquid extracted. An artificial substrate phenolphthalein phosphate (PPP) is used.
The enzyme and substrate are allowed to react in a buffer solution, the substrate being
degraded to phosphate and free phenolphthalein.
phosphatase
phenolphthalein phosphate
phenolphthalein + phosphate
This reaction will be carried out with different molarities of sodium phosphate added
to the buffer. As phosphate is a product of phosphatase activity, it may have an effect
on the activity of the enzyme. End product inhibition is a type of negative feedback
commonly used to control the rate of a metabolic pathway in living things.
After a period of incubation any free phenolphthalein formed can be detected by
adding alkali (sodium carbonate) as at this pH phenolphthalein is pink.
Acid pH
phenolphthalein colourless
sodium
carbonate
Alkaline pH
phenolphthalein pink
Thus, the more active the enzyme the more intense the pink colour. The intensity of
colour can be made quantitative using a colorimeter. The sodium carbonate also
denatures the phosphatase and stops the reaction.
Biology: Practical Activities (AH)
35
Equipment and materials
Materials required by each student/group:
mortar and pestle
filter funnel
piece of muslin (approx. 12 cm x 12 cm)
centrifuge tube
test tube
5 boiling tubes with rack
marker pen
2 x 5 cm3 syringes
2 x 1 cm3 syringes/pipettes
stirring rod
5 cm3 phenolphthalein phosphate
beaker containing at least 25 cm3 10% sodium carbonate solution
Materials to be shared:
water bath at 30C
500 cm3 pH 5 buffer
5 beakers each with an accurate 100 cm3 graduation mark
spatula
balance
weighing boats/filter paper
Sodium phosphate (NaH2PO4)
5 droppers
500g packet of beansprouts
bench centrifuge
colorimeter with cuvettes or test tubes as appropriate
550nm filter for colorimeter
stop clock
Wear gloves and eye protection whilst carrying out this experiment. The buffer
solution, the sodium phosphate and the phenolphthalein phosphate are all possible
irritants. If any of these substances come in contact with eyes, wash immediately with
plenty of water.
Biology: Practical Activities (AH)
36
Instructions
N.B. Results from different groups may be averaged. It is therefore important for all
groups to carry out the instructions in a similar manner so that other variables are not
introduced.
1. Label 5 beakers 1 - 5 and distribute amongst the class.
2. The molarities of sodium phosphate to be present in each beaker is shown in the
table. You will have to calculate the mass of sodium phosphate required in 100
cm3 of buffer to achieve these molarities. The formula weight in grams of the
sodium phosphate is 138.0. Help with this calculation is available in the
Supplementary Student Information sheet: Calculating the mass of a chemical
required to obtain a solution with a certain molarity.
beaker number
1
2
3
4
5
molarity of sodium phosphate added
to buffer
0
0.05M
0.10M
0.20M
0.30M
Make out a table showing the mass of sodium phosphate required for 100 cm3 of
each solution.
Weigh out the appropriate mass of sodium phosphate for each beaker. Add it to
the beaker along with about 90 cm3 buffer solution.
3. Stir until it is completely dissolved and then with a dropper carefully add more
buffer until the solution reaches the 100 cm3 mark.
4. Put about 20g of beansprouts in a mortar and grind to a paste using the pestle.
5. Filter the liquid through muslin into a clean centrifuge tube.
Biology: Practical Activities (AH)
37
6. Centrifuge at high speed for about five minutes.
7. Pour the liquid (the supernatant) into a clean test
tube being careful not to disturb the pellet. This
liquid will be used as the enzyme solution.
8. Collect 5 boiling tubes in a rack and label them 1 5. Using a syringe add 5 cm3 from beaker 1
(containing plain buffer) to tube 1; then using the
same syringe add 5 cm3 from beaker 2 to tube 2
and continue this same procedure step wise to
beaker 5.
9. Add 1 cm3 of the substrate, phenolphthalein
phosphate to each tube.
10. Add 1 cm3 of enzyme solution to each tube and
mix well. To avoid serious cross contamination
with the stirring rod think about the order you
stir the test tubes.
11. Incubate all tubes at 30C for 20 minutes.
Do not incubate for longer. The phosphate may
be a competitive inhibitor. This means that
given sufficient time the enzyme will break
down all the substrate in all the tubes.
12. Add 5 cm3 of 10% sodium carbonate solution to each tube and mix as before.
(Tubes can now be stored in a refrigerator until next day if required)
13. Using water as a blank, measure the intensity of the pink colour using a
colorimeter with a 550nm filter.
14. Present your results in a table with suitable headings. Present your results as a
graph with suitable scales and axis labelled with quantities and units (a line of
best fit might be more appropriate if a straight line graph is unlikely).
15. Collect results from other groups in the class. They should be very similar!
Calculate the average value of transmission or absorption for each tube.
16. Redraw the graph using the average values. For each molarity also plot the
highest and lowest values obtained by the class. Draw a vertical line from the
highest to the lowest value for each point. This will indicate the range for each
point plotted (resuts which differ markedly from the norm should be discussed
and on the results of this discussion either be included or ignored).
Biology: Practical Activities (AH)
38
Unit: Cell and Molecular Biology (AH): Molecular interactions in cell events:
Catalysis
Title: The effect of the end product, phosphate, on the enzyme phosphatase
SUPPLEMENTARY STUDENT INFORMATION
Calculating the mass of a chemical required to obtain a solution with a certain
molarity
The atoms that make up an element each have a certain mass e.g. sodium (Na) atoms
on average have an atomic mass of 22.99, chlorine (Cl) atoms a mass of 35.44. When
different atoms combine to form molecules of a compound, the formula weight of that
compound can be calculated by adding the atomic masses together.
e.g.
sodium chloride has the chemical formula NaCl
so its formula weight is 22.99 + 35.44 = 58.43
To obtain a one molar solution (1M), the formula weight in grams is dissolved and
made up to one litre with water.
e.g.
The formula weight of sodium chloride (common salt) is 58.43
To obtain a 1M solution of sodium chloride, 58.43g of it would be dissolved in
water and the solution made up to 1 litre.
If you wanted to make just 100 cm3 of a 1M solution, 5.84g of sodium chloride
would be in the solution.
If you wanted to make 100 cm3 of a 0.1M solution, only 0.58g of sodium
chloride would be required.
How much sodium chloride would you weigh out to make:
(i) 100 cm3 of a 0.2M solution?
(ii) 100 cm3 of a 0.05M solution?
(iii) 200 cm3 of a 0.1M solution?
(answers at the bottom of the page)
The chemical you are about to work with is sodium phosphate. Its formula is
NaH2PO4. H20 and its formula weight is 138.0
How much sodium phosphate would you weigh out to make:
(i) 100 cm3 of a 1M solution?
(ii) 100 cm3 of a 0.1M solution?
(iii) 100 cm3 of a 0.05M solution?
(iv) 100 cm3 of a 0.2M solution?
(v) 100 cm3 of a 10.3M solution?
Answers to sodium chloride problems (i) 1.16g (ii) 0.29g (iii) 1.16g
Biology: Practical Activities (AH)
39
Unit: Cell and Molecular Biology (AH): Applications of DNA technology
Title: Gel electrophoresis of DNA treated with restriction enzymes
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 cutting DNA with restriction enzymes
 develop problem solving skills and in particular Outcome 2 PCs:
(c) conclusions drawn are valid and explanations given are supported by evidence
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
This experiment is done with the help of Plant DNA Investigation kit obtained from
NCBE, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ.
Tel: 0118 987 3743 Fax: 0118 975 0140. Cost £130.00 (2000 prices). SAPS offers
sponsorship towards the initial cost of a kit providing that a teacher from the school
has attended a SAPS DNA workshop. Contact SAPS at Edinburgh University
(tel: 0131 650 7124) or at Head Office (tel: 01223 507168) to obtain the appropriate
form. Refills and individual items can also be obtained from NCBE. Student Guides
and Technical Guide are supplied with the kit. Pages 10-15 of the Student Guide and
the Technical Guide supply a great deal of relevant background information.
In this experiment it is assumed that a 4-tooth gel comb is used to provide 4 wells in
each gel. If using a 6-tooth gel comb the wells hold less DNA and any resulting
bands will be fainter. Four-tooth gel combs are available from NCBE (5 for £5.00).
Classroom management
This experiment requires three separate days to be completed.



Day 1 - Practising with the microsyringe and digesting the DNA requires 30-40
minutes followed by a 40 minute incubation at 37C. After the incubation the
small tubes should be stored in the refrigerator until the next day.
Day 2 - Separating the DNA fragments requires about 30 minutes to set up.
Ensure the gels are loaded close to the electricity supply so they do not have to be
moved once loaded. As long as the electric current has been applied long enough
for the DNA to have moved out of the wells (40-50 minutes) the electricity can be
switched off and on as required. A current of 9 volts will require a total of 12
hours electricity for the separation; 18 volts will require 5-6 hours of electricity in
total.
Day 3 - Staining the gels requires only 5-10 minutes but the gel can take another
15-20 minutes to identify any visible bands and measure the distance each band
has travelled.
Biology: Practical Activities (AH)
40
Supply of materials
In order to satisfy the core skill in problem solving, students will be required 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 PCs b-f.
PC 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 migrate through the gel, the rate of movement being
linked to the size of the fragment; Day 3 - the DNA is stained and the number
of base pairs in any visible band identified by referring to the table supplied.
PC c: a table of results with appropriate headings and units showing the size of each
visible DNA fragment and the distance it has travelled.
PC d: a graph of the results. It is probably best with the size of DNA fragment
(number of base pairs) on the x-axis and the distance travelled (mm) on the yaxis.
PC e: a conclusion stating that the smaller the DNA fragment the further it will travel;
however, the relationship is not linear e.g. a small fragment half the size of
another fragment will travel more than twice the distance of the larger
fragment.
PC f: evaluation points include:
 was the DNA mixed enough each time it was transferred? If too 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 and an appropriate
voltage used? DNA bands should be spaced out over the entire gel; appropriate
voltage is 1-5 volts per centimetre (the distance between the two electrodes).
 corrosion may occur at the anode; despite this, the electrophoresis should not be
effected.
 if the gel is blank then either the DNA has not been adequately rehydrated or the
stain has not been left in contact with the gel for long enough.
 why are the smaller DNA fragments not visible? What size must the fragments
in your gel be before they are visible?
 why have some fragments not separated sufficiently to be seen as separate
bands?
Biology: Practical Activities (AH)
41
References
Investigating Plant DNA - Student Guide and Technical Guide. These booklets
accompany the DNA kit available from NCBE.
Miller, M.B. (1993) DNA technology in schools: a straightforward approach,
Biotechnology Education, 4(1), 15-21.
Miller, M.B. (1994) Practical DNA technology in school, Journal of Biological
Education, 28(3) 203-211.
Miller, M.B. and Russell, G.A. (1996) Practical DNA technology in school - 2:
Computer analysis of bacteriophage lambda base sequence, Journal of Biological
Education, 30(3) 176-183.
This practical was based on work initially carried out by M.B. Miller and G.A Russell
while involved in the SAPS project.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
Biology: Practical Activities (AH)
42
Unit: Cell and Molecular Biology (AH): Applications of DNA technology
Title: Gel electrophoresis of DNA treated with restriction enzymes
TECHNICAL GUIDE
Materials required
Materials required by each student/group:
Day 1 - 2 pink tubes containing the restriction enzyme EcoR1
2 green tubes containing the restriction enzyme Hind111
1 yellow tube (empty)
1 white tube of DNA suspension
1 microsyringe and 6 tips
1 float
1 vial loading dye
1 piece of parafilm
1 marker pen
Day 2 - electrical supply of 9-18 volts
2 electric wires with crocodile clips
enzyme tubes in the float from the previous lesson
vial of loading dye
gel in a plastic tank with comb, covered in buffer solution
microsyringe and 4 tips
piece of black card
2 pieces of carbon fibre tissue
Day 3 - tank containing your gel from previous lesson
stain (10 cm3)
70% ethanol (5 cm3)
gloves
eye protection
Materials to be shared:
Day 1 - waterbath at 37C
Day 2 - bottle of TBE buffer
Biology: Practical Activities (AH)
43
Preparation of materials
Preparation of materials supplied by the kit
Rehydrating the DNA - The  DNA in the narrow white tubes provided in the Plant
DNA kit must be rehydrated with distilled water shortly before the experiment is
carried out. Follow the instructions on page 10 of the Student Guide provided with
the kit. One tube of DNA is required per group of students.
Preparing the agarose gel - If necessary, this can be done a few days before the
experiment is carried out. Follow the instructions on page 12 of the Student Guide.
One gel is required per group of students.
Two pieces of carbon fibre electrode tissue (approximately 42mm x 22mm) are
required per group. Wear gloves when handling the carbon fibre tissue.
Dilute the concentrated electrophoresis buffer 10 times with distilled water. About 35
cm3 will be required per group (11-12 cm3 to dissolve the agarose and form the gel
and the rest to cover the gel once it is set). The liquid can be reused for 3-4 ‘runs’
after which it should be discarded.
Dilute the concentrated stain for DNA with an equal volume of distilled water. About
10 cm3 of stain is required per group. This diluted stain can also be reused several
times. Wear gloves and eye protection when handling the stain.
Recipes for the various buffers and dyes used in the experiment are given on pages 10
and 11 of 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 a small hot
rod. The holes should be about 8mm in diameter. This will allow the pointed end of
the enzyme microtubes through but will hold their top end. Alternatively, the holes
can be made in a thin piece of foam e.g. camping mat.
Pieces of parafilm (about 5cm x 5cm) are required for the microsyringe exercise.
However, any non-absorbent paper e.g. benchcoat will be suitable.
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
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. A fuller account of safety is covered
on pages 6 and 7 of the Technical Guide accompanying the kit
Biology: Practical Activities (AH)
44
Unit: Cell and Molecular Biology (AH): Applications of DNA technology
Title: Gel electrophoresis of DNA treated with restriction enzymes
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity




What is the aim of the activity?
What measurements are you going to make?
Are you familiar with how the restriction enzymes act on DNA?
Are you aware of what is happening during electrophoresis?
Getting organised for experimental work
What safety measures are you required to take?
Are you familiar with the microsyringe and how to deliver a set volume using it?
Recording of data
Prepare a table with suitable headings and units to record the number of base pairs in
each identified DNA fragment and the distance it has travelled through the gel.
Evaluation
 Why are some DNA fragments not visible?
 Why have some DNA fragments not separated sufficiently to be seen as separate
bands?
 Is there evidence that the DNA was not evenly distributed in its original tube?
What can be done to prevent this?
 How long should the electric current be passed through the gel so that DNA bands
will be separated as much as possible?
 Can you account for some lanes of the gel being blank?
Biology: Practical Activities (AH)
45
Unit: Cell and Molecular Biology (AH): Applications of DNA technology
Title: Gel electrophoresis of DNA treated with restriction enzymes
STUDENT ACTIVITY GUIDE
Introduction
This experiment uses most of the basic techniques involved in genetic fingerprinting.
The DNA is digested or ‘cut up’ using restriction enzymes. The resulting fragments
of DNA are then separated into bands using an electric current and made visible by
staining.
DNA cut with
restriction
enzymes
DNA source
electric
current
DNA fragments
of varying size
DNA fragments
separated and stained
If the order of bases in the DNA used is different each time then the DNA fragments
produced each time after digestion will also be different. Thus, DNA from different
organisms (except clones) will give a unique result in this experiment - hence the term
genetic fingerprinting.
DNA from a certain bacteriophage will be used in this experiment as only one, short
chromosome is present in the organism. This will result in only a few different
fragments being formed, thus making their separation into distinct bands more likely.
Nuclear DNA from animals or plants consists of many large chromosomes. After
digestion, a very large number of fragments are formed. If all these fragments were
stained, a smear would result. To obtain distinct bands (a fingerprint) with this
complex DNA, only certain fragments are selected using probes.
The simple, bacteriophage DNA is going to be digested in 3 different ways:
- 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
Biology: Practical Activities (AH)
46
Each restriction enzyme will cut the DNA only when a certain sequence of bases
occurs e.g. the enzyme EcoR1 cuts the DNA between bases G and A only when the
sequence GAATTC is present in the DNA. The other restriction enzyme used cuts the
DNA at a different sequence of bases. Thus, each restriction enzyme is specific.
restriction enzyme EcoR1
DNA
double
helix
C
DNA cut
into
fragments
G
T
C
G
A
A
T
T
C
G
A
C
C
C
A
G
C
T
T
A
A
G
C
T
G
G
G
T
C
G
C
A
G
C
A
T
T
A
A
T
T
A
C
G
A
C
G
C
T
G
G
The number of DNA fragments formed after digestion by an enzyme will depend on
the number of times the particular sequence of bases which the enzyme acts on is
present e.g. the sequence GAATTC occurs 5 times in the bacteriophage DNA used in
this experiment. The DNA will therefore be cut into six fragments when digested by
the enzyme EcoR1.
Equipment and materials
Materials required by each student/group:
Day 1 - 2 pink tubes containing the restriction enzyme EcoR1
2 green tubes containing the restriction enzyme Hind111
1 yellow tube (empty)
1 white tube of DNA suspension
1 microsyringe and 6 tips
1 float
1 vial loading dye
1 piece of parafilm
1 marker pen
Day 2 - electrical supply of 9-18 volts
2 electric wires with crocodile clips
enzyme tubes in the float from the previous lesson
vial of loading dye
gel in a plastic tank with comb, covered in buffer solution
microsyringe and 4 tips
piece of black card
2 pieces of carbon fibre tissue
Biology: Practical Activities (AH)
47
Day 3 - tank containing your gel from previous lesson
stain (10 cm3)
70% ethanol (5 cm3)
gloves
eye protection
Materials to be shared:
Day 1 - waterbath at 37C
Day 2 - bottle of TBE buffer
Instructions
Preliminary exercise
This experiment requires you to transfer very small volumes of liquids. A
microsyringe is provided for you to do this. The tips that fit on the end of
the microsyringe have small ‘ridges’ on them. When the tip is filled to the
upper ridge 10 l will be delivered. The lower ridge is for delivering
volumes of 2 l.
10 l
2 l
Follow the hints below when using a microsyringe.
• Before loading the microsyringe, pull the plunger out a little. This gives some
extra air with which to expel the last drop of liquid.
• When emptying the microsyringe tip, hold it vertically and at eye level.
• To remove the last droplet from the tip, touch it against the inner wall of the
container.
• Do NOT touch the point of the microsyringe tip with your fingers. There are
enzymes in sweat which may contaminate and result in unwanted digestion of
DNA samples.
• A tip must only be used once to prevent any cross-contamination occurring.
Microsyringe exercise
You may find this useful to become familiar with the microsyringe.
i)
Draw in 2 l 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 l of dye and deposit it alongside the smaller drops.
iv)
Now draw all five 2 l drops into the micropipetter tip and deposit them
alongside the 10 l drop.
v)
Are the two drops the same size?
Biology: Practical Activities (AH)
48
Day 1 - Digesting the DNA
1.
Sit the 4 tubes containing restriction enzymes in the float on the bench.
2.
With a new microsyringe tip draw the DNA suspension into and out of the
microsyringe tip several times. This results in the DNA being evenly
distributed. Now transfer 20 l of DNA to each of the TWO pink tubes
containing a restriction enzyme.
3.
Again with a new tip, transfer 20 l of DNA to ONE green tube containing a
different restriction enzyme. Remember to mix the DNA thoroughly before
transferring it.
4.
Again with a new tip, transfer 20 l of DNA to an empty yellow tube. This tube
will act as a control as here the DNA will be undigested.
5.
Cap the tubes and flick the sides of the tubes with one finger until the blue
colour is evenly spread throughout the liquid.
6.
Place the float with the 4 tubes in a waterbath at 37°C
(leaving the one remaining green tube on your bench).
7.
After 10 minutes the restriction enzymes will be in
solution. This will allow you to transfer the entire
contents of one of the pink tubes to the remaining green
tube again using a NEW tip on the microsyringe.
The DNA in this green tube will now be digested by
both restriction enzymes. Mark the tube with a D - for
double digest.
8.
Flick each tube several times to mix the contents. Put the four tubes (one pink,
one unmarked green, one green marked D and one yellow) in the float back into
the waterbath to incubate at 37°C for at least another 30-40 minutes.
N.B. The tubes can be left until next lesson as the restriction enzymes will
become denatured after a few hours. To prevent further DNA breakdown, the
tubes should be stored in a refrigerator overnight.
Biology: Practical Activities (AH)
49
Day 2 - Separating the DNA fragments
1.
Remove the comb gently from the gel to expose the wells.
2.
Ensure your tank is close to your electricity supply and place a piece of black
card under it to make the wells more visible.
3.
If not already done, cover the gel with about 20 cm3 of buffer solution (to a
depth similar to that shown in the diagram below).
Buffer solutions keep the pH stable and thus prevent unwanted breakdown of
unstable molecules such as DNA.
*4.
Using a new tip, draw in 2 l of loading dye and mix this thoroughly with the
undigested DNA in the yellow tube by drawing the mixture up and down in the
tip several times.
*5.
Draw up all the contents of the tube into the microsyringe tip and load well 1 by
emptying the syringe slowly when the end of the tip is in the buffer solution and
directly above the well.
N.B. The tip does not actually need to be in the well as the dense dye will make
the DNA solution sink.
microsyringe tip
loading dye
and DNA
buffer solution
gel
6.
Repeat the last TWO steps marked * and load each well as follows:
N.B. use a NEW microsyringe tip each time.
Well 2 - DNA digested by restriction enzyme EcoR1 (pink tube)
Well 3 - DNA digested by restriction enzyme Hind111 (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 and crocodile
clips. Once the electricity is switched on the negatively charged phosphates in
the DNA are attracted to the positive electrode. So, make sure the positive
electrode is furthest AWAY form the DNA in the wells.
9.
Switch on the electricity. The voltage must not exceed 18 volts. The TBE
buffer can evaporate during electrophoresis, periodically check the depth of the
buffer and top up as required (to a depth similar to that shown in the diagram in
Step 5).
As well as helping the DNA sink into the wells, the loading dye also allows us to
judge how long the electric current should be on by moving in front of all but
the smallest DNA fragments.
Biology: Practical Activities (AH)
50
carbon fibre
well
s
buffer
solution
10.
After an appropriate time (e.g. 12 hours at 9 volts; 6 hours at 18 volts) switch
off the electricity , disconnect the crocodile clips and remove the pieces of
carbon fibre.
Biology: Practical Activities (AH)
51
Day 3 - Staining the DNA
1.
Return the buffer solution covering the gel to its original container.
2.
Pour about 10 cm3 of staining solution (Azure A) onto the surface of the gel and
leave it for at least 4 minutes.
3.
Return the stain to its original container.
4.
Wash the gel surface with about 5 cm3 of 70% ethanol for a few seconds.
5.
Pour off the ethanol and carefully rinse the gel with cold tap water 3 or 4 times.
6.
Finally, cover with water and allow the gel to ‘develop’.
If the staining solution has been used on a previous occasion you may need to repeat
the above procedure. If this is necessary allow at least 10 minutes for instruction 2.
Lanes
1
2
3
4
Purple bands of stained DNA will appear
shortly.
The smaller the fragments of DNA the
further it will have travelled through the gel.
However, the smallest fragments will also
take up less stain and may therefore be
difficult to see. Also , fragments of similar
size will move similar distances in the gel,
resulting in little separation between them.
wells
DNA
bands
largest
smallest
Below is a table showing the number and
size of DNA fragments formed during the
experiment. This is possible as the entire
base sequence of the DNA in the bacteriophage used has been worked out.
Biology: Practical Activities (AH)
loading dye
52
Lane 1
Contents
Lane 2
Lane 3
Lane 4
Undigested DNA DNA digested by DNA digested by DNA digested by
restriction
restriction
both restriction
enzyme, EcoR1
enzyme, HindIII enzymes
No. of DNA
fragments formed
No. of base pairs
in each fragment
1
48,502
6
21,226
7,421
5,804
5,643
4,878
3,530
8
23,130
9,416
6,557
4,361
2,322
2,027
564
125
13
21,226
5,148
4,973
4,268
3,530
2,027
1,904
1,584
1,375
947
831
564
125
7.
Examine your gel and try to connect the DNA fragments listed above with the
bands that have appeared in each lane. For each identifiable band measure the
distance it has travelled. Measure from the bottom of each well to the front end
of each band.
8.
Make a table with appropriate headings and units showing the number of base
pairs and the distance travelled for each band.
9.
Present your results as a graph with suitable scales and axes labelled with
quantities and units.
Biology: Practical Activities (AH)
53
Unit: Environmental Biology (AH): Symbiotic relationships (Parasitism)
Title: Isolating and examining cysts of potato cyst nematodes
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 parasitism and more specifically of the
relationship between potato cyst nematodes (PCNs) and potato plants
 develop problem solving skills and in particular Outcome 2 PCs:
(b) information is accurately processed using calculations where appropriate
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
An outline of the life cycle, transmission and control of the potato cyst eelworm
(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 PCNs is expensive, complicated and an ever increasing problem.
There are two species of PCNs; Globodera rostochiensis and Globodera pallida.
Although both are troublesome, G. pallida is the more serious pest and becoming
increasingly difficult to control. Some varieties of potato 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 are
resistant to G. rostochiensis and partially resistant to G. pallida..
Classroom management
Obtaining suitable soil samples is covered in the Technical Guide. The initial
extraction of PCNs using sieves should take only 15-20 minutes. However, filtering
the water/soil mixture must be completed before proceeding to the next stage of the
experiment. The filtering will take about 30 minutes and, of course, longer if the
water/soil mixture is filtered a second time.
Ideally the moist filter papers should be kept overnight in a humid environment. The
cysts will then burst more readily. However, it is possible to complete the entire
experiment on the same day if necessary, although cyst bursting may be less
successful.
Biology: Practical Activities (AH)
54
Examination of the cysts will take 30-60 minutes. The filter papers are first examined
under a low power binocular microscope (x10 - x20). Cysts are transferred to a
microscope slide and then burst whilst viewing under a compound microscope (x100).
Identifying PCN cysts and distinguishing between viable and non-viable PCNs is
covered in the Student Activity Guide.
N.B. Holding PCNs normally requires a license, as it is a serious pest of a common
food crop. It is therefore important that good laboratory practice is followed at all
times during this procedure. This includes autoclaving all possible sources of viable
cysts once the experiment is completed. All possible precautions should also be
followed to prevent soil infected with viable cysts from being washed down the sink,
especially if sludge from local sewage treatment plants is spread on agricultural soil.
Care must also be taken to avoid cross-contamination of samples.
Supply of materials
In order to satisfy the core skill in problem solving, students will be required 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 PCs b-f.
PC b: a description of the method used to extract PCNs from a soil sample; a
description of a viable and non-viable PCN.
PC c: a table with suitable headings showing the total number of viable and nonviable cysts per 100g of at least two soil samples
PC d: a table with suitable headings and / or bar chart showing the % viable cysts in
at least two soil samples
PC e: a conclusion on how suitable each soil would be for producing a crop 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
Biology: Practical Activities (AH)
55
Extension work
Make exudates from resistant and non-resistant potatoes. Mix these with viable cysts
and note any differences in number of PCNs released from cysts. A method for making
exudate and inducing hatching of cysts is included in the Technical Guide.
As above but vary the exudate e.g. temperature of mixing, previously boiled, vary pH
and concentration.
Examine a variety of soils for PCNs.
Test the efficiency of the extraction method by adding a known number of cysts to a soil
sample, follow the method given and calculate the % recovered. The extraction method
can be varied and % cysts recovered monitored.
References
Atkinson H. (1997) The worm in the root!, Biological Sciences Review, 9(5), 36-38.
Evans K. A., Harling R. & Dubickas A. (1998) Application of a PCR-based technique
to speciate potato cyst nematodes and determine the distribution of Globodera pallida
in ware growing areas. Aspects of Applied 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, ISBN 0 85199 2749.
Acknowledgements
The original protocol for this experiment was obtained from the Scottish Agricultural
College (SAC), West Mains Road, Edinburgh. This information and advice from A.
Evans and C. Kasperak of the SAC are gratefully 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 Science Agency (SASA),
East Craigs, Edinburgh.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
Biology: Practical Activities (AH)
56
Unit: Environmental Biology (AH): Symbiotic relationships (Parasitism)
Title: Isolating and examining cysts of potato cyst nematodes
TECHNICAL GUIDE
Materials required
Materials required by each student/group:
large filter paper (185 mm diameter)
set of compasses with pencil
ruler
filter funnel (top internal diameter about 100 mm)
washing bottle
glass rod
large beaker e.g. 400 cm3
binocular microscope (x10 - x20)
compound microscope (x100)
piece of acetate
large conical flask e.g. 250 cm3
pair of fine forceps
microscope slides
coverslips
Materials to be shared:
dried soil, gently crushed or rolled
balance
weighing boats
soil sieves with large mesh (550 m - 850 m) - mesh no. 30 or 20
soil sieves with small mesh (250 m) - mesh no. 60
Preparation of materials
Obtaining a suitable soil sample containing viable PCNs may present a problem in
some areas. A garden or allotment with a history of growing susceptible varieties of
potatoes (see Teacher/Lecturer Guide) is usually a good source. In rural areas a local
farmer may be willing to provide suitable soil.
If taking soil samples from any land you must ensure that all equipment used and
boots worn are clean and could not be contaminated with cysts from a prior sampling
site. The distribution of cysts is unlikely to be uniform. ‘Hot spots’ will occur and so
it is important to take several samples of about 100g at intervals throughout the field.
Sampling points should be chosen randomly and small soil samples lifted using a
trowel or the widest cork borer (no. 6 - each bore will give about a 10 g sample).
SAPS may be able to supply a limited number of non-viable cysts.
Biology: Practical Activities (AH)
57
Soil samples should be dried at room temperature before use. This increases the
chances of PCN cysts floating during their extraction from soil. If the soil is not fine,
it may also need to be passed through a riddle 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 of 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 need to be stored at 4C for 3-6 months before they will hatch.
Disposal of materials
It is most important that good laboratory practice is carried out during this
experiment. All materials containing cysts must be autoclaved or soaked in bleach
before being disposed. Suitable precautions are listed in the Student Activity Guide.
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
Biology: Practical Activities (AH)
58
Unit: Environmental Biology (AH): Symbiotic relationships (Parasitism)
Title: Isolating and examining cysts of potato cyst nematodes
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity





What is the aim of the activity?
What measurement are you going to take?
Are you aware of the size of potato cyst eelworm cysts and what they look like?
Are you aware of the differences between viable and non-viable potato cyst
eelworms?
Are you aware of the precautions you must follow to prevent further spread of this
parasite?
Getting organised for experimental work
What safety measures are you required to take?
In your group decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active part in carrying out
the experiment and in collecting results.
Recording of data
Prepare a table to record
 the total number of cysts in each soil sample
 the % viable cysts
You should use a ruler, correct headings and appropriate units when necessary.
Evaluation
 Are there possible flaws in the extraction process where PCNs can be lost from
the sample, leading to unreliable results?
 Do you think the procedure involved in taking the soil sample is reliable?
 Is the sample size (50 g) large enough? (A 500 g sample is used when this
procedure is carried out professionally)
 Has the filter paper been examined sufficiently or is it possible that cysts on it
could be overlooked?
Biology: Practical Activities (AH)
59
Unit: Environmental Biology (AH): Symbiotic relationships (Parasitism)
Title: Isolating and examining cysts of potato cyst nematodes
STUDENT ACTIVITY GUIDE
Introduction
Potato cyst nematodes (PCNs), also known as potato cyst eelworms (PCEs), are
world-wide parasites of potato plants. They originated in South America where the
Incas practised a seven-course rotation to control them. Being parasites, the PCNs
receive all their nutritional requirements from the potato plant, resulting in reduced
root and foliar growth and a reduction in tuber yield. The cost of damage caused by
PCNs is estimated to be between £20-30 million each year in the UK alone. This
annual cost is increasing as is the incidence of PCNs.
Like many parasites, PCNs have a highly specialised life cycle. The cysts you are
going to isolate are only about 0.5 mm in diameter and contain 200-600 eggs which
have larvae coiled up inside them.
Cyst - light brown in colour.
Contains 200-600 eggs.
Can remain dormant in soil
for up to 30 years.
Egg containing
coiled up larva.
0.5 mm
every year a small number of
eggs are released
spontaneously. This number
increases when a susceptible
potato variety is grown in
infected soil.
Female becomes attached to
potato plant. When fertilised by
male its body swells
and develops into a cyst.
0.5 mm
Larva emerges from egg, invades root
and removes materials from both xylem
and phloem. If no host plant is
available, the larva die within days.
Biology: Practical Activities (AH)
60
Infection of potato plants by PCNs has several effects:
(i) High infection rates of PCNs will reduce yields and may result in complete crop
loss.
(ii) The plants are more susceptible to drought.
(iii) Secondary invaders e.g. fungi can enter the root system more readily.
(iv) The eelworms may be vectors for pathogenic plant viruses.
Larvae possess limited powers of movement. PCNs are thus transmitted passively in
soil by machinery, potatoes used for propagation i.e. seed potatoes, flood water and
irrigation.
They are mainly controlled by using a combination of the following:
(i) Crop rotation
(ii) Resistant varieties
(iii) A type of pesticide known as nematicides.
Equipment and materials
Materials required by each student/group:
large filter paper (185 mm diameter)
set of compasses with pencil
ruler
filter funnel (top internal diameter about 100 mm)
washing bottle
glass rod
large beaker e.g. 400 cm3
binocular microscope (x10 - x20)
compound microscope (x100)
piece of acetate
large conical flask e.g. 250 cm3
pair of fine forceps
microscope slides
coverslips
Materials to be shared:
dried soil, gently crushed or rolled
balance
weighing boats
soil sieves with large mesh (550 m - 850 m) - mesh no. 30 or 20
soil sieves with small mesh (250 m) - mesh no. 60
detergent with dropper
Biology: Practical Activities (AH)
61
Precautions required to be taken
As potato cyst nematodes are a serious pest to an economically important food crop,
the precautions listed below must be followed.
1. If taking soil samples from any land you must ensure that all equipment used and
boots worn are clean and could not be contaminated with cysts from a prior
sampling site.
2. Find out if sludge from your local sewage treatment plant is spread on agricultural
soil. If so, all possible precautions should be followed to prevent viable cysts
from being washed down the sink.
3. After use, all apparatus e.g. sieves, glassware should be autoclaved 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 previously DRIED in
the soil at room temperature.
1. Weigh out 50 g of the dried soil. The soil sample has a history of being used for
growing potatoes. Break up any small lumps GENTLY with the end of a glass rod.
2. Collect the two soil sieves, fitting the one with the larger mesh size on top. Place
the sieves above a bucket or polythene bag and add the 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 pass through the finer
sieve so it can be washed on its own under the tap. When washing the larger mesh
sieve always place the finer mesh sieve beneath it.
In the instructions that follow, treat the contents of each sieve separately. Each
group of students should therefore form two smaller groups, one working with the
soil in the large mesh sieve, the other with the soil in the small mesh sieve.
Biology: Practical Activities (AH)
62
5. Away from the sink, wash out the contents
of your allocated sieve into a beaker with
the help of a wash bottle.
To do this, hold the sieve almost at right
angles above the beaker and with a wash
bottle project a stream of water on to what
was the lower side of the sieve. Slowly
rotate the sieve while doing this. Then,
turning it the right way up , wash final
contents from the sieve. DO NOT now
wash sieves in the sink - see precautions.
sieve
soil sample
wash
bottle
6. Allow the soil/water mixture to settle until
little movement of material is occurring
(10 minutes).
15 mm
7.
Using a pair of compasses and a pencil
draw four concentric circles on a large filter
paper, as shown in the diagram . Ensure the
circles drawn are complete and prominent.
Draw a straight line from the centre to the edge
of the filter paper .
185 mm
8.
Fold this filter paper twice and fit it into
a filter funnel. Sit the funnel on top of a large
conical flask.
9. Once the contents of the beaker have settled, decant quickly into the filter paper
without disturbing the sunken soil. While decanting, rotate the beaker slowly so
that any floating debris stuck to the sides gets washed into the filter paper.
10. Add a drop of detergent to the soil/water mixture while it is filtering. This
encourages any cysts present to migrate to the sides and stick to the paper.
11. Using a high pressure flow of water add about 200 cm3 to the beaker containing
the soil. Allow to settle and decant as before into the filter paper.
12. Once filtration is complete remove the filter paper from the funnel, 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. a piece of
acetate) and examine under the binocular microscope. Starting at the straight line
in the outermost circle, examine this circle for cysts. Repeat this procedure for
the other circles on the filter paper.
Biology: Practical Activities (AH)
63
Potato cyst eelworm cysts are only 0.5 mm in diameter on average.
However, they are easily detected by their shape and colour perfectly spherical apart from a small ‘neck’ (rather like a gourd or
spherical decorations commonly put on Christmas trees). They
vary from being orange and copper coloured to a dull dark brown.
14. With a pair of fine forceps remove any cysts from the filter paper and place in a
droplet of water on a microscope slide. The concentric circles drawn previously
should help to ensure the entire filter paper is scanned although most cysts should
be found in the outermost circle. Count the total number of cysts found on the
filter paper. Add this to the number found on the filter paper from the other sieve
of the same soil sample.
15. Select at random several cysts and place them far enough apart on a few
microscope slides so that each can be covered by a separate coverslip. Add a
drop of water to each cyst and cover each one with a coverslip.
16. Examine each cyst in turn under a microscope (x100 total magnification). Whilst
viewing a cyst press down gently on the coverslip. 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 rectangular objects as in the
diagram of the life cycle. Determine the number of cysts containing viable
larvae.
N.B. Do not attempt to burst open all the eggs. a cyst just needs to contain ONE
viable larvae for it to be scored as viable. If cysts are completely empty, assume
they are non-viable.
Viable larvae will uncoil
completely when the egg case
bursts. Their ‘skin’ will be smooth
and free of any sudden
indentations.
non-viable larvae will have folds
and ‘kinks’ in their ‘skin’.
16. Calculate
i.
the total number of cysts per 100 g of your soil sample (you started with a 50 g
sample).
ii.
the % viable cysts in your random sample of cysts
18. Compare the soil sample you have just examined with one with a different history
for growing potatoes.
19. Present your results in a table with suitable headings. Draw a bar chart with the
axes labelled appropriately to show the results graphically.
Biology: Practical Activities (AH)
64
The experiment you have just done is a simplified, scaled-down version of a test
carried out routinely on fields aimed to produce seed potatoes. If even one viable
potato cyst eelworm is found in a 500g sample then the field cannot be used to
provide seed potatoes.
N.B.
1) any results obtained form this experiment are for guidance only and do
NOT have the status of an official test.
2) soil and any equipment used in the experiment must now be autoclaved to
kill any PCN cysts. Do NOT dispose of any soil samples by returning them to
land from which they did not originate.
Biology: Practical Activities (AH)
65
Unit: Environmental Biology (AH): Interactions in ecosystems: Symbiotic
relationships (Mutualism)
Title: Examining mycorrhizae and the environmental factors affecting their
abundance
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 mutualism and in particular, those
benefits obtained from a mycorrhizal relationship
 develop problem solving skills and in particular Outcome 2 PCs:
(b) information is accurately processed using calculations where appropriate
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
A basic description of mycorrhizae, the benefits a plant enjoys by having a
mycorrhiza and possible commercial uses, are all outlined in the Student Activity
Guide.
If required, the references listed on page 2 can provide a fuller background.
Classroom management
Mycorrhizae are seasonal. Roots are best collected from March to June. Advice on
collecting roots is in the Technical Guide. Roots of the same species of plant are
required from TWO different locations. Again, refer to Technical Guide for suitable
locations.
Students can work individually or in pairs for this experiment.
Preparing and staining the roots takes about one hour although much of this time is
waiting for the roots to clear. Once stained and in water, preparing and examining the
roots to establish the frequency of mycorrhization may take a further 30 minutes.
This experiment would be eminently suitable for carrying out during a field trip in
May and June.
Supply of materials
In order to satisfy the core skill in problem solving, students will be required to
identify and obtain resources required for themselves. Further advice on supply of
material is given in the Technical Guide.
Biology: Practical Activities (AH)
66
Advice on marking Outcome 3 report
Specific advice for PCs b-f.
PC b: to include the different habitats of the collected roots; ‘clearing’ of roots,
staining and microscopic examination.
PC c: a table of results with appropriate headings showing the degree of
mycorrhization in fifteen 1 cm lengths of roots from two different habitats.
PC d: the frequency of mycorrhization for the two sets of roots has been calculated
correctly.
PC e: a conclusion is made connecting, if possible, the difference in habitat and the
degree of mycorrhization e.g. the higher the moisture level the lower the
incidence of a mycorrhiza.
PC f: evaluation points include:
 have the roots been collected, washed and stored correctly and at the right time
of year?
 have enough plants in each habitat been studied for a meaningful conclusion to
be made?
 have roots been ‘cleared’ the right amount (if clearing is insufficient the root
cortex cells will contain cytoplasm and the fungal hyphae will not be as
prominent, if clearing has been too severe the root cortex will be missing or
broken)?
 is the ‘infection rate chart’ sufficiently accurate?
Extension work
N.B. In the field, mycorrhizae are best collected from March to June.






Examine a variety of plant roots and compare the intensities of mycorrhizal
infection. Generally, the greater the presence of a highly branched network of fine
roots the less need for a mycorrhizal relationship.
Identify a species with a good mycorrhizal infection in several plots. Apply a high
phosphorous fertiliser to half the plots periodically. After several weeks, compare
mycorrhizal frequencies in the differently treated plots.
Investigate the effect of high moisture levels on mycorrhizal frequency e.g.
compare a sloping area with a flat area at the bottom of the slope.
Compare mycorrhizal frequencies in lawns or woods of different ages. A
mycorrhiza can take a number of years to develop.
Compare mycorrhizal frequencies in cultivated and uncultivated land.
Investigate the frequency of a mycorrhiza in a chosen species throughout its
growing season.
Biology: Practical Activities (AH)
67
Reference
De Mars B.G. and Boerner R.E.J. (1995) A simple method for observing vesiculararbuscular mycorrhizae with suggestions for designing class activities. Journal of
Biological Education, 29 (3), 209-214.
Grace C. and Stribley D.P. (1991) A safer procedure for routine staining of vesiculararbuscular mycorrhizal fungi. Mycological Research , 95 (10), 1160-1162.
Web Site:
http://helios.bto.ed.ac.uk/bto/microbes/mycorrh.htm
Acknowledgements
The original protocol for this experiment was obtained from the Scottish Agricultural
College (SAC), West Mains Road, Edinburgh. Information and advice from Dr L.
Harrier is gratefully acknowledged. Information and advice was also received from
Dr P.A. Mason, Institute of Terrestrial Ecology, Bush Estate, Penicuik, Midlothian.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
Biology: Practical Activities (AH)
68
Unit: Environmental Biology (AH): Interactions in ecosystems: Symbiotic
relationships (Mutualism)
Title: Examining mycorrhizae and the environmental factors affecting their
abundance
TECHNICAL GUIDE
Materials required
Materials required by each student/group:
compound microscope (x100, x400 magnification)
binocular microscope (x10-x50 magnification)
scissors
dropping bottle 50% glycerol
2 modified 20 cm3 syringes (see below)
forceps
2 petri dishes
stopclock
gloves
eye protection
marker pen
Materials to be shared
beaker containing 10% potassium hydroxide
beaker containing 1% hydrochloric acid
beaker containing 0.1% methyl blue in 50% lactic acid
water bath at 80C
two batches of same species of roots from different environments each group of
students will require a minimum of 15 cm of roots from each batch (see below for
guidance on collection, preparation and storage of roots).
Preparation of materials
Wear gloves and eye protection when preparing the 10% potassium hydroxide, 1%
hydrochloric acid and the stain (0.1% methyl blue in 50% lactic acid).
Methyl blue is available from Philip Harris, cat. no. S55750/4, £3.26 for 5g.
Alternatively a 1% aqueous solution, cat. no. S55760/7, £2.93 for 100 cm3 can be
used.
Lactic acid is available from Fisher Scientific UK Limited, Bishop Meadow Road,
Loughborough, Leicestershire LE11 0RG (Tel: 01509 231166) cat. no. CAS 598-823, £16.70 for 500 cm3.
To prepare 100 cm3 of stain, dissolve 0.1 g methyl blue in 50 cm3 of water and add 50
cm3 lactic acid. Aniline blue can be used instead of methyl blue.
Biology: Practical Activities (AH)
69
To make the modified syringes: Remove the nozzle end of a 20 cm3 syringe barrel
with a sharp knife or hacksaw to make the barrel into an open tube. Heat this cut end
briefly in a bunsen flame. Carry out in a fume cupboard or well-ventilated room. Do
NOT melt the plastic. Immediately press this hot, cut end onto a piece of nylon mesh.
The mesh will stick to the end of the syringe barrel, trim around the stuck edge with
scissors. A suitable mesh is available from the SAPS Office (tel: 0131 650 7124).
This modified syringe is useful for bringing the roots in contact safely with the
various chemicals involved in the experiment.
Collection, Preparation and storage of roots
Mycorrhizae are seasonal. They are most abundant in spring and early summer and
may even be absent or only in spore form when roots are dormant.
It is estimated that 90% of herbaceous plants can form mycorrhizal associations. It
should therefore, in theory, be easy to obtain appropriate root samples. Ajuga repens
(Bugle) and Glechoma hederacea (ground ivy) are particularly suitable. Avoid
members of the Brassica family, e.g. cabbage, and Chenopodia families, e.g.
goosefoot, fat hen, as these rarely, if ever, form mycorrhizae. In March the roots of
Galanthus nivalis (snowdrop) from gardens have a high incidence of mycorrhiza.
N.B. Before collecting plants ensure that you obtain the landowners permission to do
so. Be careful not to remove protected plant species from the wild.
When collecting plants, consider the following points:
i. Leave as much soil as possible sticking to the roots
ii. If not being washed immediately, store in a plastic bag in a refrigerator. Do not
let the roots dry out. Do not freeze.
iii. When ready, soak roots in warm water to remove soil.
iv. Avoid larger roots; collect intermediate and small roots.
v. Once washed, store the roots in 70-95% alcohol if not being stained immediately.
The plant roots collected should be of the same species and come from two
environments, ideally varying in just one factor. Where possible, the environmental
difference should be measured and noted. e.g.
i. a soil with low moisture level (on a slope) and a soil with higher moisture level
(at the bottom of the slope). Use a soil moisture meter and record readings.
ii. two similar habitats of different ages (an established lawn and a recently laid one;
an old wood and a more recently planted one). Note the ages of the lawns/woods.
iii. the same type of plant in the same habitat but roots collected in different months
during the growing season. Label each batch of roots with the date collected.
iv. a plot treated with a high phosphorus fertiliser and a similar untreated plot. Note
the date(s) of application and the NPK values of the fertiliser.
Biology: Practical Activities (AH)
70
Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
Biology: Practical Activities (AH)
71
Unit: Environmental Biology (AH): Interactions in ecosystems: Symbiotic
relationships (Mutualism)
Title: Examining mycorrhizae and the environmental factors affecting their
abundance
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity
 What is the aim of the activity?
 Two batches of roots of the same plant species are available. Do you know what
environmental condition was different between the two batches? If possible,
obtain quantitative measurements of this variable.
 What measurements are you going to make?
Getting organised for experimental work
 What safety measures are you required to take?
 Familiarise yourself with the modified syringes.
 Have you previously used a binocular microscope?
 Can you successfully examine material under a microscope at x400
magnification?
 In your group decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active part in setting up
the experiment and in collecting results.
Recording of data
 Prepare a table to record the degree of mycorrhization for each batch of roots.
 A table showing frequency of mycorrhization and the environmental factor being
varied should also be prepared.
 You should use a ruler, correct headings and appropriate units.
Evaluation
 Has the ‘clearing’ procedure of roots been carried out long enough? (any fungal
hyphae present are clearly visible in the root cortex if ‘clearing’ of root cells has
been carried out successfully. If ‘clearing’ has been carried out for too long a
time, or the potassium hydroxide has been too concentrated, the root cortex
becomes disintegrated.
 Has the stain been taken up sufficiently by the fungal hyphae? (The potassium
hydroxide used in ‘clearing’ must be neutralised by the washing in water and
hydrochloric acid before the stain will be absorbed efficiently).
 Is the infection rate chart reliable enough to use?
 Has the environmental difference between the root batches altered the frequency
of mycorrhization?
 Is any difference in mycorrhization between the two batches significant?
 The stain used does not differentiate between mutualistic and pathogenic fungi.
What precautions could you take during sampling to ensure that the roots did
indeed contain mutualistic fungi?
Biology: Practical Activities (AH)
72
Unit: Environmental Biology (AH): Interactions in ecosystems: Symbiotic
relationships (Mutualism)
Title: Examining mycorrhizae and the environmental factors affecting their
abundance
STUDENT ACTIVITY GUIDE
Introduction
Mycorrhizae are mutualistic associations between fungi and the roots of higher plants.
There are two major types: ectomycorrhizae and endomycorrhizae (often referred to
as arbuscular mycorrhizae). In ectomycorrhizae the fungus surrounds the host root,
whereas in endomycorrhizae the fungus penetrates the host root cells. Some 90% of
plant species commonly form endomycorrhizae.
Both the fungus and plant benefit from the association. The fungus benefits by
receiving carbohydrates (produced by photosynthesis) from the plant. The plant in
return receives minerals (especially phosphorous) from the fungal hyphae. Other
benefits plants may obtain by a mycorrhizal association are:
1. increased tolerance to drought
2. increased resistance to some soil pathogens
3. rapid growth at the beginning of the growing season
4. some plants cannot achieve reproductive maturity without a mycorrhizal
association
Studies have also revealed possible commercial uses:
1. inoculating tree seedlings with appropriate mycorrhizal fungi increases the
chances of successful regeneration of a forest when the seedlings are planted out.
2. mycorrhizae increase metal or acid uptake in plants and can therefore be used to
reduce the level of toxicity in soils.
In this practical you will work with two batches of roots. Each batch will be from the
same species. Ideally, only one environmental factor will be different in the habitats
used for sampling e.g. soil moisture, phosphate level, age of ecosystem.
Alternatively, time of year the roots were collected could be the variable.
Find out how the environment varied between the two root samples. You are going to
stain roots of a particular species taken from each sample, examine for mycorrhizae
and estimate their abundance. A conclusion on how the environmental variable
affects mycorrhizal abundance can then be made.
Biology: Practical Activities (AH)
73
Equipment and materials
Materials required by each student/group:
compound microscope (x100, x400 magnification)
binocular microscope (x10-x50 magnification)
scissors
dropping bottle of 50% glycerol
microscope slides
coverslips
2 modified 20 cm3 syringes
forceps
2 petri dishes
stopclock
gloves
eye protection
marker pen
Materials to be shared:
beakers containing 10% potassium hydroxide
beakers containing 1% hydrochloric acid
beakers containing 0.1% methyl blue in 50% lactic acid
two batches of same species of roots from different environments
water bath at 80C
Instructions
1.
Collect the two samples of roots. The roots are from the same species of plant.
Your teacher/lecturer will tell you how their environments varied. You must
treat the two batches of roots separately and label any apparatus they are in
appropriately.
2.
Wash the roots in tap water and collect 5-6 root strands from each sample.
3.
Cut one lot of roots into about 1 cm lengths, remove plunger from modified
syringe and place roots for staining into syringe barrel. Replace plunger and
push down to about the 5 cm3 mark. Treat other batch of roots similarly.
4.
Place syringes in beaker containing 10% potassium hydroxide and draw up
about 10 cm3 of solution into syringe. Incubate beaker and its contents in a
waterbath at 80C for about 45 minutes.
This liquid is corrosive and a strong alkali. Wear gloves and eye protection
when using it.
Potassium hydroxide solution removes the cytoplasm from the root cells. This
step is therefore usually referred to as ‘clearing’.
Biology: Practical Activities (AH)
74
5.
By drawing liquids in and out of the syringe, rinse roots in (i) water
(ii) 1% hydrochloric acid.
Potassium hydroxide must be neutralised for stain to work efficiently.
6.
Draw the stain (0.1% methyl blue in 50% lactic acid) into the syringe for at least
5 minutes at room temperature.
The lactic acid is also corrosive. Continue to wear eye protection and gloves.
This treatment stains the hyphae a bright blue or even purple. Hyphae are long
thin strands of fungi. Some root tissues e.g. vascular bundles, stain paler blue.
Roots can be stored in this stain or simply in water for several months.
7.
Rinse in water as before.
8.
Remove roots from syringe barrel and immerse in a little water in a petri dish.
Examine roots under a binocular microscope. Look for signs of mycorrhizal
infection - the root cortex will contain bright blue or purple staining hyphal
threads.
9.
Mount fifteen 1 cm lengths of root onto a microscope slide in 50% glycerol.
10.
Place a coverslip on top of the root pieces.
11.
While examining under the microscope, determine the degree of infection in
each root piece. Consult the Infection rate chart (below) while carrying out this
step. Make out a table with appropriate headings for these results.
12.
Calculate the frequency of mycorrhization using the following formula:
F% = 100 (N - No)
N
where N is the number of root pieces examined
No is the number of root pieces with only a trace or no
mycorrhiza
13.
Compare the frequency of mycorrhization in each sample of roots. If possible,
make a hypothesis as to how the environmental difference between the two root
batches affects the degree of mycorrhization.
Biology: Practical Activities (AH)
75
Infection rate chart
Class:
0
1
2
3
4
5
where 0 = no infection
1 = trace infection
2 = 10% of root piece infected
3 = between 11% and 50% of root piece infected
4 = between 51% and 90% of root piece infected
5 = over 90% of root piece infected
Biology: Practical Activities (AH)
76
Unit: Environmental Biology (AH): Interactions in ecosystems: Degrees of
interaction
Title: The effect of relative humidity on the development of Botrytis infection in
plants
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 how environmental factors influence
interaction between species
 develop problem solving skills and in particular Outcome 2 PCs:
(c) conclusions drawn are valid and explanations given are supported by evidence
(d) experimental procedures are planned, designed and evaluated appropriately.
Background information
The experiment involves incubating raspberries at various humidities. The raspberries
have been inoculated with the fungal pathogen Botrytis cinerea. The SAPS ELISA kit
can then be used to quantify the severity of the Botrytis infection in each case. A link
between humidity and development of Botrytis infection can then be made.
The SAPS ELISA kit can be obtained from Robert Burns, Scottish Agricultural
Science Agency (SASA), Monoclonal Antibody Unit, 2 Craigs Road, East Craigs,
Edinburgh EH12 8NJ.
Tel: 0131 244 8911
Fax: 0131 244 8987
Email: burns@sasa.gov.uk
Final cost of the kit will be £30 + VAT.
The kit contains enough materials for at least 5 ELISA runs and 5 copies of a Student
Guide and 1 copy of a Technical Guide. Sufficient background information,
including references, is covered in the Guides.
Classroom management
Ideally, the experiment should be completed in one session of about 2 hours. If this is
unsuitable, then once the wells contain the filtrates they can be covered with cling
film and left overnight in the refrigerator.
Supply of materials
In order to satisfy the core skill in problem solving, students will be required to
identify and obtain resources required for themselves. Further advice on supply of
material is given in the Technical Guide.
Biology: Practical Activities (AH)
77
Advice on marking Outcome 3 report
Specific advice for PCs b-f.
PC b: a description of the preparation of the filtrates; the various molecules trapped
in the wells and how they combine with one another; the role of the PBST
PC c: a table with suitable headings showing the relative humidity and the relative
Botrytis units present in each well
PC d: a line graph with % relative humidity on the x-axis and Botrytis units on the yaxis (It may be necessary to plot the log of Botrytis units as the units on the
colour chart show a geometric progression).
PC e: a conclusion stating the relationship between % humidity and concentration of
Botrytis in the fruit
PC f: evaluation points include:
 how effective PBST has been in preventing the antibodies from binding to the
walls of the wells. (If the control well is blue then the PBST is faulty or the
wells are not being washed out correctly. PBST contains detergent. It is used to
wash out wells to prevent molecules sticking to the well walls.)
 there may be difficulty in estimating Botrytis concentration accurately using the
colour chart provided. A more precise estimate may be obtained using a light
probe.
 the kit has a shelf life of only 2-3 months. Also, if the secondary antibody is not
stored at 0-4C, it may separate from the enzyme. This will result in none of the
wells turning blue.
 using the Pastette provided in the kit, the volumes of filtrate, monoclonal
antibody, secondary antibody and substrate can all be accurately controlled.
This is necessary for meaningful comparisons between the wells.
 the raspberries have been inoculated in the same way
 results of different groups have been compared to show possible variations
arising from differences in the raspberries used at each humidity e.g. ripeness,
size, innate infections
 has the % relative humidity in the various jars been constant over the 2-3 days
while the infected raspberries have been present (the humidity may well rise
over the 2-3 days as the raspberries give off water vapour)
 the concentration of Botrytis in each well must be read at the same time after
adding the substrate. The blue colour intensifies with time in all the wells.
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Extension work
Suitable extension work is covered in the SAPS Guides accompanying the kit.
A possibility not covered in the Guides: Inoculate raspberries with another microorganism e.g. Saccharomyces cerevisiae, Bacillus subtilis. Once the initial infection
is established, inoculate with Botrytis. Using suitable controls, it may be possible to
detect some degree of biological control taking place i.e. the initial micro-organism
may prevent the Botrytis infecting the raspberry.
Acknowledgements
Information and advice from Dr Molly Dewey, Department of Plant Sciences,
University of Oxford and Dr Mary MacDonald, Biogemma (UK) Ltd., Cambridge, is
gratefully acknowledged. Information and advice was also received from Robert
Burns, SASA, Edinburgh.
This experiment was produced by the SAPS Biotechnology Scotland Project.
Funding for the project was provided by SAPS, Unilever and The Scottish Office.
Support was also provided by Edinburgh University, Quest International, the Scottish
CCC, the Higher Still Development Unit and SSERC.
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Unit: Environmental Biology (AH): Interactions in ecosystems: Degrees of
interaction
Title: The effect of relative humidity on the development of Botrytis infection in
plants
TECHNICAL GUIDE
Preparation of Raspberries
N.B. This preparation requires to be started 5-7 days before the experiment is
carried out.
Day 1: - Remove raspberries from freezer (if possible select raspberries of equal size
and ripeness and with no obvious signs of infection).
- Place on a bed of paper towels to soak up excess moisture, and cover. If
necessary the raspberries can be stored like this in the refrigerator over the
weekend.
Day 2: - Add about 20 cm3 water to one storage jar and label it “Botrytis at high
humidity”.
Add about 5g of silica gel to another storage jar and label it “Botrytis at
medium humidity”.
Add about 50g of silica gel to the third storage jar and label it “Botrytis at
low humidity”.
If possible, adjust the readings in the relative humidity meters so they all
register the same reading (about 30%) and sellotape one to the inside of each
jar.
- Collect 3 small beakers, put 2 raspberries into each beaker and then put 1
beaker into each of the jars. Ensure the lid of the jar is tightly sealed.
Day 3: - Check the humidities in the jars cover a wide range e.g. 85%, 50%, 25%
- renew the silica gel in the medium (5g) and low (50g) humidity jars (any
silica gel which has turned pale blue/pink can be recycled if placed in a hot
oven overnight).
- Use forceps to lift out each raspberry in turn and roll it gently over the
surface of a Botrytis culture. Return each raspberry to the jar it came from.
- Leave the sealed jars in a warm room for 2-3 days. This will allow Botrytis
to grow if the conditions are favourable.
Preparation of Solutions
PBS (phosphate buffered saline) - Dissolve each tablet in 200 cm3 of distilled or clean
tap water. Set aside about 40 cm3 for filling the small plastic tubes labelled PBS.
PBST (phosphate buffered saline with Tween 20) - Use a 1 cm3 pipette from the kit to
add 2 drops of Tween to the remaining volume (about 160 cm3) of PBS. Transfer 30
cm3 to each large plastic tube labelled PBST.
Biology: Practical Activities (AH)
80
Monoclonal antibody (Mab) - Using a 1 cm3 pipette from the kit transfer 0.5 cm3 to
each, appropriately labelled, microcup.
Secondary antibody - enzyme conjugate (Ab-EC) - A very small volume (10l) of
the concentrate is provided in each of the 5 screw capped Eppendorf tubes labelled
‘Ab-EC’. Using a 1 cm3 pipette from the kit, add 1 cm3 PBST to each tube and agitate
well to mix. Using the same 1 cm3 pipette, dispense 0.5 cm3 into each, appropriately
labelled, microcup.
For further background and safety precautions please refer to the Guides
accompanying the SAPS ELISA kit.
Materials required
Materials required by each student/group:
supplied from the kit
4 small disposable plastic tubes - labelled as follows:
- PBS (containing at least 6.5 cm3 of PBS)
- FF from LH (fruit filtrate from low humidity)
- FF from MH (fruit filtrate from medium humidity)
- FF from HH (fruit filtrate from high humidity)
1 large disposable plastic tube (contains 30 cm3 PBST)
3 x 1 cm3 pipettes
5 Pastettes
4 microwells
2 microcups - labelled as follows:
- MAb (contains 0.5 cm3 monoclonal antibody)
- Ab-EC (contains 0.5 cm3 second antibody-enzyme conjugate)
3 muslin squares
supplied by the school/college
1 pair forceps
3 glass rods
3 clean test tubes (or small plastic tubes supplied in the kit, if possible)
1 container for waste
gloves may be worn by students with sensitive skin
Materials to be shared:
Cultures of the fungus Botrytis cinerea (supplied in kit)
6 raspberries (from punnet of frozen raspberries)
3 storage jars with a tight seal (at least 500 cm3 capacity)
e.g. from Philip Harris - catalogue number Y 61960/9 - pack of 12 @ £9.29
3 relative humidity meters
e.g. from Philip Harris - catalogue number K 67850/1 - £6.52 each.
Remove mounting bracket when using jars of above size.
silica gel (dark blue in colour)
1 bottle of TMB (supplied in kit)
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Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray
system for each student/group. Equipment and materials should be supplied in a way
that students have to identify and obtain resources. Normal laboratory apparatus
should not be made available in kits but should generally be available in the
laboratory. Trays could be provided containing one type of specialist equipment or
materials.
Disposal of materials
Any unused antibodies should be autoclaved or bleach added to them before being
binned.
All other apparatus from the kit (e.g. plastic tubes, Pastettes, microwells, microcups)
must be binned after use and not reused.
Other apparatus contaminated with Botrytis can be washed in hot soapy water (it is
not thought necessary to autoclave as Botrytis spores are extremely common and not a
human pathogen).
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82
Unit: Environmental Biology (AH): Interactions in ecosystems: Degrees of
interaction
Title: The effect of relative humidity on the development of Botrytis infection in
plants
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and background information and safety
precautions in the Student Guide provided with the SAPS ELISA kit. Consider the
following questions.
Analysis of activity
 What is the aim of the activity?
 What measurements are you required to take? Is the time you take them crucial?
 Although what is happening in each well is invisible, can you imagine what is
taking place at each step in the procedure?
 Are you aware of the background involved in the preparation of both the
monoclonal antibody and the secondary antibody enzyme conjugate?
 Do you know why the initial solutions in the wells are mixed with PBS buffer
while subsequent solutions are mixed with PBST?
 Do you know the importance of the washing procedure between each step and
how to carry it out?
Getting organised for experimental work
 What safety measures are you required to take?
 Are you familiar with the Pastettes used to deliver drops of a set volume?
 In your group decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active part in setting up
the experiment and in collecting results.
Recording of data
Prepare a table to record:
i. the different relative humidities in which the raspberries were inoculated.
ii. the relative Botrytis concentration in each well.
You should use a ruler, correct headings and appropriate units when necessary.
Evaluation
 Is the control well colourless? If not, can you explain how a blue colour may
result here?
 Are the colours in the colour chart sufficiently true to estimate the Botrytis
concentrations accurately for each well?
 Are there some variables that have been difficult to control e.g. the raspberries
used? If some of these have affected the results what can be done to minimalise
the effect?
 Was the intensity of blue colour in each well measured at the same time after
adding the substrate? If not, how might this affect the results?
 Was the humidity in each jar more or less constant over the 2 day incubation
period?
Biology: Practical Activities (AH)
83
Unit: Environmental Biology (AH): Interactions in ecosystems: Degrees of
interaction
Title: The effect of relative humidity on the development of Botrytis infection in
plants
STUDENT ACTIVITY GUIDE
Introduction
Raspberries inoculated with the fungal pathogen Botrytis have been incubated for 2-3
days at different humidities. An ELISA kit is available which allows the severity of a
Botrytis infection to be determined. Thus, a possible link between humidity and the
development of Botrytis can be investigated.
The ELISA technique is used widely. Some possible uses are listed below:
a) To detect traces of certain food components e.g. peanuts for a person with a food
allergy
b) To diagnose infectious diseases in humans e.g. HIV, herpes, Legionnaires disease.
c) To diagnose plant diseases, e.g. to ensure vegetatively propagated plants such as
potatoes are disease-free, or to detect specific diseases such as the fungal disease
Botrytis (as used in the SAPS ELISA kit) even before the plant shows visible
signs of infection.
d) To determine the level of a drug (or any drug metabolite) or a hormone in the
blood, e.g. to find out if an athlete has been taking performance-enhancing
substances.
e) To determine if a gene has been successfully transferred in a genetic modification
experiment (the protein that the transferred gene codes for will be detected).
ELISA is an acronym for:
ENZYME LINKED IMMUNO-SORBENT ASSAY
i.e. A substance can be detected (assay), even in extremely low concentrations, by it
sticking to a surface (sorbent). Then, the specificity between antigens and antibodies
(immuno) used to combine a specially made monoclonal antibody to the stuck
substance. A second antibody is added which combines with the monoclonal
antibody. The second antibody has an enzyme attached to it (enzyme linked). A
colourless substrate is then added, which the enzyme changes to a coloured product.
The intensity of the colour formed indicates how much of the initial substance was
present in the sample tested.
A background of how the monoclonal antibody was prepared is covered in the SAPS
ELISA Student Guide.
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Equipment and materials
Materials required by each student/group:
supplied from the kit
4 small disposable plastic tubes - labelled as follows:
- PBS (containing at least 6.5 cm3 of PBS)
- FF from LH (fruit filtrate from low humidity)
- FF from MH (fruit filtrate from medium humidity)
- FF from HH (fruit filtrate from high humidity)
1 large disposable plastic tube (contains 30 cm3 PBST)
3 x 1 cm3 pipettes
5 Pastettes
4 microwells
2 microcups - labelled as follows:
- MAb (contains 0.5 cm3 monoclonal antibody)
- Ab-EC (contains 0.5 cm3 second antibody-enzyme conjugate)
3 muslin squares
supplied by the school/college
1 pair forceps
3 glass rods
1 container for waste
3 clean test tubes
Materials to be shared:
3 sealed jars, at different humidities, containing raspberries inoculated with Botrytis
cinerea
1 bottle of TMB (supplied in kit)
gloves
Instructions
You will set up your tests in 4 microwells, designated 1, 2, 3, 4 as follows:
1 - PBS only (acts as control)
2 - fruit filtrate from raspberry incubated in low humidity
3 - fruit filtrate from raspberry incubated in medium humidity
4 - fruit filtrate from raspberry incubated in high humidity
Biology: Practical Activities (AH)
85
Preparing the fruit filtrates for diagnosis of Botrytis
All the raspberries in each jar were inoculated with Botrytis cinerea 2-3 days ago and
then kept at the different humidities as shown by the relative humidity meter in each
jar. Note each humidity.
1. Use clean forceps to transfer about a quarter of a raspberry from the jar labelled
“Botrytis at low humidity” into a clean test tube (or ideally a small plastic tube
from the kit if sufficient have been provided).
If the piece of raspberry is too large many molecules from it will bind to the well
walls instead of the Botrytis antigen.
2. Use a 1 cm3 pipette to transfer 2 cm3 of PBS into the tube.
3. Use a glass rod to gently break up the piece of fruit to form a pulp (this may take
several minutes as the raspberry will be hard and dry).
Repeat instructions 1-3 using a quarter of a raspberry incubated at medium humidity
and another at high humidity. Label the tubes appropriately.
4. Make the liquid levels in the three tubes the same by adding an appropriate volume
of distilled water to the first and second tubes. Filter each pulp separately through
moist muslin into a suitably labelled disposable plastic tube e.g. FF from LH (fruit
filtrate from low humidity).
Coating the wells
5. Label the microwells 1, 2, 3 and 4 with a marker pen.
6. Use a clean Pastette to transfer 4 drops of PBS into well 1.
7. Use the same Pastette to transfer 4 drops of fruit filtrate from low humidity into
well 2.
8. Use a clean Pastette to transfer 4 drops of fruit filtrate from medium humidity into
well 3.
9.
Use a clean Pastette to transfer 4 drops of fruit filtrate from high humidity into
well 4.
Discard used Pastettes and other apparatus into the waste container provided.
Leave for at least 10 minutes.
During this time many different
antigens, including the Botrytis
antigen, if present, become
attached to the walls of the
wells.
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86
10. Empty all 4 wells by inverting them above the waste container or sink. Remove
the last drops by tapping the wells upside down on a pad of paper towel then
wash all wells thoroughly (3 times) with PBST.
For each washing use a 1 cm3 pipette to fill the wells with PBST. Empty each
well, then fill again with PBST. Repeat 3 times, removing all buffer each time.
Wells may be tapped upside down on a pad of the paper towel to remove
remaining droplets of liquid (do not worry if there are bubbles at the bottom of
the wells - these disappear when the next reagent is added). After the last wash it
is important to ensure that no liquid remains. Keep the pipette for dispensing
PBST in steps 12 and 14.
11. Use a clean Pastette to add 4 drops of the MAb to each well.
Leave for at least 10 minutes.
At this stage, although many
antigens are present, the
specially made monoclonal
antibody (MAb) will bind only
with the Botrytis antigen.
12. Empty all 4 wells by inverting them above the waste container or sink. Remove
the last drops by tapping the wells upside down on a pad of paper towel. Wash
each well 3 times with PBST (see step 10).
13. Use a clean Pastette to add 4 drops of the Ab-EC to each well.
Leave for at least 20 minutes.
The second antibody enzyme
conjugate (Ab-EC) is designed
so that it will attach itself only to
the monoclonal antibody.
14. Empty all 4 wells by inverting them above the waste container or sink. Remove
the last drops by tapping the wells upside down on a pad of paper towel. Wash
each well 3 times with PBST (see step 10).
15. Add 4 drops of TMB liquid substrate solution (from the dropper bottle) to each
well. Wait for the colour to develop.
Biology: Practical Activities (AH)
87
TMB liquid substrate solution is an irritant and toxic. You should therefore avoid
contact with skin and eyes.
The enzyme attached to the second
antibody changes the colourless
TMB substrate to a blue colour.
The darker the blue colour the more
Botrytis antigen was present in the
original sample.
The colour should be visible within 5 minutes but may take up to 30 minutes to
develop fully.
16. Using the colour chart, try to estimate the incidence of the Botrytis infection at the
same time for each well as the intensity of the blue colour increases with time.
The numbers on the colour chart refer to relative units of Botrytis. Note that the
concentration doubles with each increase in the colour intensity.
17. Compare the incidence of the Botrytis infection with the different humidities in
which the raspberries were incubated.
18. Present your results in a table with suitable headings. Draw a graph of the results
with the axes labelled appropriately. To allow results to be spread evenly it may be
necessary to plot the ‘log of Botrytis infection’ for one axis.
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Appendix 1
PREPARING FOR THE ACTIVITY
Read through the Student Activity Guide and consider the following questions.
Analysis of activity
 What is the aim of the activity?
 What is being varied in the activity?
 What variables must be kept constant?
 What measurements/observations are you going to make?
 What controls are present in the experimental design and why?
Getting organised for experimental work
What safety measures are you required to take?
In your groups decide how the activity will be managed by allocating tasks to each
member. For Outcome 3 it is important that you play an active part in setting up the
experiment and in collecting results.
Recording of data
Prepare a table to record the results. You should use a ruler, correct headings and
appropriate units.
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Outcome 3: Advice to Candidates
Writing a report on an experiment in Biology
Appendix 2
This advice is designed to help you write a report to meet the performance criteria of Outcome 3. You
must have played an active part in 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 the microscope’ say ‘slides were examined under the microscope’.
Always use the past tense in writing reports. Write your report using the following headings and pay
attention to the advice under each heading.
It is useful to structure your report under specific headings to avoid missing out important sections. The
following headings can be used:
Title
Check
Use the title in the Student Activity Guide.
Aim
A brief statement of the purpose of the experiment.

Method
Write a brief description of how the experiment was carried out. Do not put in too much detail, just
sufficient so that anyone reading your report would know what you did rather than be able to repeat the
experiment exactly. 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.

Results
Record your raw data in a clear table with correct headings, appropriate units and results/readings
entered correctly.

Analysis and presentation of results
You should analyse and present your results using one or more of the following:
 a table with suitable headings and units, showing averages or other appropriate computations
 a graph presented as a histogram, bar chart, connected points, line of best fit as appropriate,
with suitable scales and axes labelled with quantity and units, and with data correctly plotted
 a scatter diagram or equivalent.

Conclusion
Your conclusion should use evidence from your experiment and relate back to the aim of the
experiment. You should include, at least one of the following,:
 overall pattern to readings or observations (raw data)
 trends in analysed information or results
 connection between variables and/or controls.

Evaluation
The evaluation could cover all stages of the activity including preparing for the activity, analysis of the
activity, and the results of the activity. Your evaluation must include supporting argument in at least
one of the following:
 effectiveness of procedures
 control of variables
 limitations of equipment
 possible sources of error
 possible improvements.
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Outcome 3: Teacher/Lecturer Guide: Biology
Appendix 3
All the performance criteria given in the left-hand column must be achieved in order to attain the
outcome. The right-hand column gives suggestions which might aid the professional judgment of the
assessor.
PERFORMANCE CRITERIA
SUGGESTIONS TO AID PROFESSIONAL JUDGMENT
a.
The information is collected
by active participation in the
experiment
The candidate has taken active part in the collection of the
information.
b.
The experimental procedures
are described accurately.
A clear statement of the aim of the experiment.
A few brief concise sentences including as appropriate:

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. The use of the
impersonal passive voice is to be encouraged as an example of
good practice but this is not mandatory for meeting the
performance criteria.
c.
Relevant measurements and
observations are recorded in
an appropriate format.
Readings or observations (raw data) must be recorded in a clear
table with correct headings, appropriate units and results/readings
entered correctly.
d.
Recorded information is
analysed and presented in an
appropriate format
Data should be analysed and presented in tabular, graphical
format or as a scatter diagram or equivalent as appropriate:
e.
Conclusions drawn are valid.

For a tabular presentation this may be an extension of the
table used for performance criteria c. above, and must
include: suitable headings and units showing averages or
other appropriate 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
quantity and units and with data correctly plotted.
Conclusions should use evidence from the experiment and relate
back to the aim of the experiment. At least one of the following
should be included:



f.
The experimental procedures
are evaluated with
supporting argument.
The evaluation could cover all stages of the activity including
preparing for the activity, analysis of the activity, and the results
of the activity. The evaluation must include supporting argument
in at least one of the following.





Biology: Practical Activities (AH)
overall pattern to readings or observations (raw data)
trends in analysed information or results
connection between variables and/or controls.
effectiveness of procedures
control of variables
limitations of equipment
possible sources of error
possible improvements.
91
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