AS Biology
Experiment Book
Name:................................................................................................................
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Prepared October 2013 by C. Coetzer
Includes;
Practical 1 - Investigation into size and scale of microscopic tissues
Practical 2 - Microscopic observation of cells and tissues
Practical 3 – Mitosis and root tip squash (Exam Question)
Food Tests
Practical 4 - The qualitative analysis of sugars (ie detecting their presence or absence)
Practical 5 -Testing for the presence of sugar in fizzy drinks
Practical 6 - Food test detective work.
Practical 7 - The action of saliva on starch
Practical 8 - Estimate the concentration of reducing sugar in a fruit juice (Exam question)
Practical 9 – Testing different food stuffs
Practical 10 - The identification of biological chemicals present in solutions
Practical 11 – Identify starch, protein and reducing sugar in various solutions
Practical 12 - Diffusion in liquid
Practical 13 – Diffusion in agar blocks (Exam Question)
Practical 14 - Investigating Osmosis
Practical 15 – Effects of different salt solutions on potato chips (Exam Question)
Practical 16 - Plasmolysis
Practical 17 - The effect of temperature on membrane permeability in beetroot
Practical 18 - Investigating the action of the enzyme Catalase
Practical 19 - Investigating the effect of substrate concentration on the rate of an enzyme
controlled reaction.
Practical 20 – Yeast and its enzymes (Exam question)
Practical 21 – Enzyme hydrolysis of starch to glucose (Exam question)
Practical 22 - The effect of wind speed on the rate of transpiration in a leafy shoot
Practical 23 - Potometer Practical
Practical 24 - Stomatal Density in Agapanthus
Practical 25- Pathways for gases in a leaf
Practical 26 - Vital capacity and tidal volume
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Prepared October 2013 by C. Coetzer
Practical 1 - Investigation into size and scale of microscopic tissues
This practical focuses on microscope technique and using graticules and stage micrometers to determine size
and scale in biological cells and tissues.
Intended learning outcomes
By the end of this practical you should be able to:
• Use a microscope fitted with an eyepiece graticule and stage micrometer
• Calibrate the eyepiece graticule using the stage micrometer
• Use the calibrated graticule to determine the actual size of microscopic specimens
• estimate the accuracy of a measurement
• Use the graticule to determine scales
• Understand the importance of repeating or validating set of results.
Background information
• The measurement of specimen size with a microscope, is made by using an eyepiece graticule. This is a
glass or plastic disc with 8 divisions etched onto its surface, which is inserted into the eyepiece lens.
• The size of the eyepiece graticule remains constant, despite the fact that the image viewed will change its
size depending upon whether high- or low-power objective lenses are used. For example a cell viewed with the
x40 objective will appear much larger than when viewed with the x10 objective. However because the graticule
is in the eyepiece it will not change its size. Therefore the value of each of the divisions in the eyepiece
graticule varies with the magnification of the objective lens.
• A stage micrometer is a very accurately etched glass or plastic ruler that is placed on the microscope stage so
that the eyepiece graticule scale is superimposed on the stage micrometer scale. The scale is usually 1mm
divided into 100 separate divisions so that each division equals 10 micrometres (10μm).
• It is necessary to calibrate the eyepiece graticule with the stage micrometer placed on the microscope stage
for each objective lens used.
You will observe a TS of plant tissues through a microscope and use an eyepiece graticule and a stage
micrometer to determine the size of some of the structures.
• Read the information above.
• Ensure that you understand the principles of using an eyepiece graticule and a stage micrometer before you
continue with the investigation.
Method
Preparation
1. You have been provided with a compound light microscope with both lowand high-power objective lenses
and an eyepiece lens that has been fitted with a graticule. You have also been provided with a stage
micrometer.
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Prepared October 2013 by C. Coetzer
2. You must now calibrate the eyepiece graticule.
Place the stage micrometer onto the microscope stage and focus using the low-power objective lens so that the
graticule scale becomes superimposed over the stage micrometer scale.
3. Move the stage micrometer until the start or zero line of each scale is coincident (lined up)
4. Look along the scale until another coincident point is found.
5. The relationship between the two scales can now be calculated
On the scale shown there are 17 divisions on the stage micrometer scale that line up with 7 divisions on the
graticule scale.
Thus 17 / 7 = 2.42857 units.
Each unit on the stage micrometer scale is 10 micrometres (10μm).
Therefore each division on the graticule scale is 24.2857 micrometres rounded to 24.3 μm.
6. Use the procedure described above to determine the size of each division on the eyepiece graticule using
the low-power objective lens of your microscope.
7. Repeat the procedure to determine the size of each division when using the high-power objective lens.
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Prepared October 2013 by C. Coetzer
Making observations
1. You are provided with a stained transverse section through part of a dicotyledonous plant root.
2. Examine the specimen using the low-power of your microscope.
3. Make a large, plan drawing to show the distribution of tissues, labelling the stele (vascular bundle).
4. Use the eyepiece graticule to measure the width of the vascular bundle at its widest point in graticule units
and then calculate the actual width of the vascular bundle in millimetres and in micrometres.
5. Draw a straight line on your drawing across the vascular bundle to show where you took your measurement.
Write the dimension on your drawing next to the line.
6. Make a high-power drawing to show a group of four xylem vessels from inside the vascular bundle.
7. Use the eyepiece graticule to measure the width of the xylem vessel at its widest point in graticule units and
then calculate the actual width of the vessel in micrometres, remembering to use the appropriate calibration of
the eyepiece graticule for the high-power objective lens.
8. Draw a straight line on your drawing across the xylem vessel to show where you took your measurement.
Write the dimension on your drawing next to the line.
9. Look at your two measurements and check on their accuracy. The actual size of the xylem vessel should be
smaller than the size of the vascular bundle even though it looked larger using the high power objective lens.
10. You are now going to determine the magnification of your drawing of the xylem vessels. Use a ruler to
measure the length of the line that you drew across the xylem vessel.
Use your knowledge of the actual size of the vessel to calculate the magnification of your drawing. Write your
answer x at the bottom right hand corner of your drawing.
Follow-up
• Compare your results with other members of the class and check for consistency of readings.
• Did any member of the class have anomalous results? What are the potential causes of such an anomalous
result in this investigation?
• Write up your procedure including a discussion of the benefits of comparing your results with other students.
Practical 2 - Microscopic observation of cells and tissues
This practical focuses on microscope technique and making and recording observations in the form of
biological drawings.
Intended learning outcomes
By the end of this practical you should be able to:
• Use a microscope with skill and precision
• Show all the structures that can be seen in the defined part of a specimen
• Make clear, accurate, labelled, scale drawings of specimens
Background information
• Drawings should be done with a sharp HB pencil making clear single lines.
Examiners do not give credit for sketchy lined drawings. A soft rubber can be used to correct errors.
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• Always draw what you see and not what you expect to see from memory or textbook diagrams.
• Candidates often draw diagrams too small but rarely draw them too large.
Ensure that your drawing is large enough to show all the detail.
• All parts of the drawing should be kept in correct proportions. In poor quality drawings, proportions changes
as the drawing progresses.
• Biological drawings can be both high-power and low-power.
• Low-power drawings are usually plan drawings that do not contain cellular detail but do show the distribution
of various tissues. When a plan drawing is requested, examiners may give credit for not drawing cellular detail.
• If more than one drawing of the same or different specimens or parts of a specimen are made, examiners may
ask that they are drawn to the same scale (which means the same magnification). Credit is then awarded for
this skill.
• Look at the following two sets of drawings of a red and white blood cell, made by different students and how
marks would be allocated by an examiner.
Student A would be awarded 1 mark. Student B would be awarded 6 marks.
You will observe a TS of plant tissues through a microscope using both low and high power and draw
appropriate structures. Read the information above.
• Read your textbook and look carefully at any drawings that have been made or biological material. Take care
however; the quality of drawings in some textbooks is not all that could be desired.
• Write down the key features that are found in good biological drawings.
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Prepared October 2013 by C. Coetzer
Method - Preparation
1. You have been provided with a compound light microscope with both lowand high-power objective lenses
and a slide of a TS of a plant stem.
2. Place the slide onto the stage of the microscope.
3. Adjust the light source so that you can see a bright light when looking through the eyepiece lens.
4. With some microscopes it is possible to rack the objective lens so far down that it will break the slide. In
order to prevent this it is good microscope technique to:
• set the objective lens on low power.
• not look through the eyepiece but to look at the side of the microscope and carefully lower the objective lens
until it is nearly, but not quite touching the slide.
• now look through the eyepiece and gradually raise the objective lens until the slide comes into focus.
5. You should now carefully move the slide around on the stage until you find the area that you wish to observe.
6. To change to high power, do not re-focus, but change the objective lens from low to high power. The slide
should be almost in focus and only a fine adjustment to the focus should be necessary.
7. Practice focussing the slide on both low and high power until you are familiar with the technique.
Making observations
1. You are provided with a stained transverse section through part of a dicotyledonous plant.
Examine the specimen using the low-power of your microscope.
Make a large, labelled, plan drawing to show the distribution of tissues.
2. Make a high power drawing to show a group of four cells from the region nearest the centre of the specimen.
Follow-up
• State from which part of the plant the section was taken. Explain your answer
• Exchange your drawings with another student and mark their drawings using the following mark scheme.
Mark scheme
Plan drawing
Corner vascular bundles larger than other vascular bundles √
No individual cells drawn √
Four sided shape to plan √
Both xylem and phloem correctly labelled √
Parenchyma correctly labelled √
Sclerenchyma on outer edge of vascular bundle labelled √
Collenchyma in corners labelled √
High power drawing
Good quality of drawing i.e. clear single lines √
4 cells only drawn, similar in size and shape √
between 5-8 sides to each cell √
Air spaces shown between corners of cells √
Thin cell walls shown either by a thin single line or two lines close together √
• Add up the marks out of 12 and return the drawings to the student.
• Write a list of all the reasons why you did not score full marks with your own drawings.
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Prepared October 2013 by C. Coetzer
Practical 3 – Mitosis and root tip squash (Exam Question)
R1 is a slide of a stained longitudinal section through a root tip.
You are also provided with an eyepiece graticule that has been fitted to the eyepiece of your microscope and a
stage scale (stage micrometer) printed on acetate sheet.
The 1 cm stage scale is divided into 100 divisions.
Each division is 0.1 mm
(a) (i) Draw a low-power plan diagram of R1 and show the area in which you can see cells undergoing mitosis.
[2]
(ii) Carefully examine this area using the high-power of your microscope. Identify a cell that has a clear nucleus
but is not undergoing mitosis.
Count the number of divisions of the eyepiece graticule across the cell.
number of divisions ......................................
Remove the slide R1 and replace it with the stage scale.
Using the same magnification, adjust the focus until you can see the eyepiece graticule on top of the stage
scale.
Count the number of eyepiece graticule divisions that match an exact number of stage scale divisions.
number of eyepiece graticule divisions ......................................
number of stage scale divisions ......................................
Use this information to calculate the actual width of the cell.
Show your working.
actual width of cell ......................................
[2]
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(iii) Estimate the uncertainty in the measurement of the actual width of the cell.
uncertainty = ......................................
[1]
(iv) Suggest how an error in measuring the lengths could occur.
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(b) Fig. 3.1 is a photomicrograph showing cells from further up the root.
Fig 3.1
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(i) Prepare the space below so that it is suitable for you to compare and contrast a cell like the one you
measured in (a) (ii) with the cells in Fig. 3.1.
Record your observations in the space below.
[4]
(ii) Roots grow in length.
State one observation from the materials provided that confirms this and suggest how such growth occurs.
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(c) Cells undergoing mitosis can be seen in the specimen on slide R1.
In the space below, make a high-power labelled, drawing of two cells showing chromosomes, undergoing
different stages of mitosis.
[5]
[Total : 17]
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Prepared October 2013 by C. Coetzer
Food Tests
Reducing Sugars include all monosaccharides, Such as glucose and fructose, and some disaccharides, such
as maltose. Use 0.1 – 1% sugar solutions.
Test for reducing sugar:
Benedicts test: Add 2 cm3 of a solution of the reducing sugar to a test tube. Add an equal volume of benedicts
solution. Shake and bring gently to the boil, shaking continuously to minimise spitting.
Observation: The initial blue coloration of the mixture turns green, then yellowish and may finally form a brick
red precipitate.
Basis of test: Benedicts solution contains copper sulphate. Reducing sugars reduce soluble blue copper
sulphate containing copper (II) ions (Cu2+) to insoluble red - brown copper oxide containing copper (I). The
latter is seen as a precipitate.
Non- Reducing sugars, the most common non reducing sugars is sucrose, a disacchriade. If reducing sugars
have been shown to be absent (negative result in a above test) a brick red precipitate in the test below
indicates the presence of a non reducing sugar. If reducing sugars have been shown to be present, a heavier
precipitate will be observed in the following test than with the reducing test if non reducing sugar is also
present.
Tests for non reducing sugar:
Add 2 cm3 of sucrose solution to a test tube. Add 1 cm3 dilute Hydrochloric acid. Boil for 1 minute.
Carefully neutralise with sodium hydrogencarbonate (Check with pH paper) Care is required because
effervescence occurs. Carry out benedicts test.
Observation: As benedicts test.
Basis of test: Disaccharide can be hydrolysed to it’s monosaccharade constituents by boiling with dilute
hydrochloric acid. Sucrose is hydrolysed to glucose and fructose, both of which are reducing sugars and give
the reducing sugar result with the benedicts test.
Test for starch: Iodine/potassium iodide test.
Add 2cm3 of 1%starch solution to a test tube. Add a few drops of iodine potassium solution.
Alternatively add the latter to the solid for of starch.
Observation: A blue-black colouration.
Basis of test: A polyiodide complex is formed with starch.
Test for a lipid:
Emulsion test: Add 2cm3 fat or oil to a test tube containing 2cm3 of absolute ethanol. Dissolve the lipid by
shaking vigorously. Add an equal amount of cold water.
Observation: A cloudy white suspension.
Basis of test: Lipids are immiscible with water. Adding water to a solution of the lipid in alcohol results in
emulsion of tiny droplets in the water which reflect light and give a white , opalescent appearance.
Test for a protein:
Biuret test: Add 2cm3 protein solution to a test tube. Add an equal volume of 5% potassium hydroxide solution
and mix. Add 2 drops of 1% copper sulphate solution and mix. No heating is required.
Observation: A mauve or purple colour develops slowly.
Basis of test: A test for peptide bonds. In the presence of dilute copper sulphate in alkaline solution, nitrogen
atoms in the peptide chain for a purple complex with copper(II) ions (Cu2+). Buiret is a compound derived from
urea which also contains the –COHN- group and gives positive results.
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Prepared October 2013 by C. Coetzer
Practical 4 - The qualitative analysis of sugars (ie detecting their presence or absence)

You have solutions of 4 sugars: glucose, fructose, lactose and sucrose.

Glucose and fructose are both hexose monosaccharides, lactose and sucrose are disaccharides.
1. Write out the formula of the monosaccharides and calculate the mass of each sugar you would need to
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make up 250cm of each solution. Explain how you would do this accurately.
2. Repeat for the lactose (a disaccharide of glucose and galactose) and sucrose (a disaccharide of glucose
and fructose)
Sugars can be classified as being either a reducing sugar or a non-reducing sugar, depending on their
2+
+
ability to reduce soluble blue Cu ions to insoluble Cu ions that is seen as an orange precipitate.
The relative concentration of sugar affects the mass of precipitate that appears, ie it is proportional and also the
time taken for the precipitate to appear (a precipitate will appear more quickly in a concentrated sugar solution
than in a dilute solution)
Testing for a reducing sugar. SAFETY GOGGLES MUST BE WORN AND HAIR TIED BACK. THIS IS NOT
NEGOTIABLE

Put 2cm of each sugar solution into a test-tube,

Label the tube, writing the contents on the top of the tube with a waterproof pen

add 2cm of Benedict’s solution and

shake to mix the contents of the tube.

Place this tube in a beaker of boiling water (the depth of the water must be higher than the contents of the
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tube)

You may put all 4 tubes in the beaker at the same time.

Record the time taken for a precipitate to appear.
Sugar
Time /s for orange
precipitate to appear
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Testing for a non-reducing sugar
If an orange precipitate does not appear then the sugar is not a reducing sugar.

3
You must now hydrolyse the sugar (start again with a fresh sample) by boiling 2cm of the sugar with 2 cm
3
of hydrochloric acid for I minute. (This is called acid hydrolysis, it splits the disaccharide into 2
monosaccharide molecules)

SAFETY: If you spill any acid mop it up immediately with a paper towel and put it in the bin. If you spill it on
your hands, wash it off with plenty of running water,

3
Cool the tube under running cold water then slowly add about 2cm of sodium hydrogen carbonate solution
to neutralise the acid.

Repeat the Benedict’s test.

If an orange precipitate appears then the original solution was a non-reducing sugar.
Practical 5 -Testing for the presence of sugar in fizzy drinks
The presence of sugar can be tested using a reagent called Benedict’s Solution.
Equipment and Reagents
Bunsen Burner and stand, 3 test-tubes, pen, pipettes, 250 ml beakers of water, glucose solution, Fizzy drinks
(normal and lite) and Benedict’s Solution.
Method
1. Put the water in a 250 ml beaker to boil.
2. Put about 1 cm depth of the glucose, 7Up and 7Up lite in each of three test tubes.
3. Add the same depth of Benedict’s solution.
4. Label each solution.
5. Place the three test-tubes into the beaker of boiling water.
6. Observe solutions, record the colours and appearance (eg clear or cloudy or presence of a precipitate)
of the test-tubes before and after boiling, and identify 7 Up.
Results
Solution
Colour before boiling
Colour after boiling
Glucose
7 Up
7 Up Free
Conclusion
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Practical 6 - Food test detective work.
You are supplied with samples of urine from three different patients. A doctor suggests that one of these is from
a person with diabetes and one from a person with a kidney problem that lets protein into the urine. She is not
sure about the third sample. Unfortunately she did not record which sample was from which patient.
Perform a test for Reducing sugar AND protein on EACH of the three samples.
Record your observations and conclusions in the tables below.
(Remember that your conclusion should just state whether protein/reducing sugar is present/absent)
Benedict’s Test for reducing sugar
Urine sample
A
B
C
A
B
C
Appearance before heating
Appearance after heating
Conclusion
Biuret test for Protein
Urine sample
Appearance before test
Appearance after test
Conclusion
Practical 7 - The action of saliva on starch
Saliva is produced in the salivary glands. It acts as a lubricant to allow food to be swallowed more easily. It also
contains an enzyme called salivary amylase that acts on cooked starch (a very long, insoluble molecule) to
break it down into smaller, soluble molecules of maltose.
Hypothesis: Saliva contains an enzyme that breaks down starch.
Apparatus and method.

3
Collect some saliva (20 cm is sufficient) by chewing on a clean rubber band (this stimulates salivation) and
spitting into a small beaker.

Put the thick starch suspension into 3 test-tubes until each is about ¾ full.

Add 5cm of the saliva to one tube, 5cm of boiled and cooled saliva to another, and 5cm of water to the
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3
3
third tube. Make sure there is about 1cm of air at the top

Mix the contents of each tube thoroughly by stirring or inverting several times and place them in a test-tube
rack (or stand them in a beaker of water at 30°C)
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
Every 5 minutes add one drop of each mixture on to a tile and add 2 drops of iodine solution to the drop
and observe the colour. If starch is still present the iodine solution will turn black.

When the tube with unboiled saliva fails to turn the iodine solution black, pour about 1 cm depth into
another test-tube, add 1 cm depth of Benedict’s solution and place the tube into a beaker of boiling water.
Repeat this with the other 2 tubes.
Results
Contents of tube. Starch +
Time /min to fail to turn iodine black
Colour when boiled with Benedict’s soln
Saliva
Boiled and cooled saliva
Water
Conclusions
1. Explain why (a) water and (b) boiled saliva were added to 2 tubes.
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2. Explain the colour changes in each tube after boiling with Benedict’s solution.
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3. Explain why the same depths of liquids had to be put into each tube.
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4. Suggest 2 ways that you could change the method to improve on the accuracy of this investigation.
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5. Did your results support your hypothesis? Explain your answer
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Practical 8 - Estimate the concentration of reducing sugar in a fruit juice (Exam
question)
You are required to estimate the concentration of reducing sugar in a fruit juice, labelled F1, by comparison with
that in a range of glucose solutions.
You are provided with a 0.8% solution of glucose, labelled F2, Benedict’s solution, distilled water and five testtubes.
(a) Carry out the Benedict’s test on fruit juice F1.
Describe and explain your results.
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(b) You are now going to test a range of glucose solutions that you will prepare yourself using F2 and distilled
water.
Carefully follow the instructions below.
You should present and record your observations and data in the space provided.
You will need to:
• read through the instructions carefully
• prepare the space on the next page so that it is ready for you to record the readings
• decide how many different strength glucose solutions you will need and prepare these by serial dilution
• carry out the tests so that you can compare your results with the result for the fruit juice.
(i) Record the data in the space below.
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(ii) Estimate the concentration of reducing sugar in the fruit juice F1.
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[1]
(c) The volume of reactants can affect the results that you obtain.
(i) State how you controlled this variable in your investigation.
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(ii) Identify two other significant sources of error in this experiment.
1...............................................................................................................................................................................
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2...............................................................................................................................................................................
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(d) Suggest how the student could improve this experiment.
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(e) A student used another carbohydrate, starch, to investigate the effect of pH on the activity of the enzyme
amylase.
The data in Table 8.1 were obtained.
Table 8.1
(i) When the student first performed this investigation, the time taken for complete hydrolysis at pH 7 was 17
minutes.
Explain why the student discarded this result and repeated the experiment with freshly made solutions.
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(ii) Complete Table 8.1 by calculating the missing mean time value for pH 9.
You may use the space below to show your working.
Put your answer in the space on Table 8.1.
[1]
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(iii) Plot a graph to show the effect of pH on the complete hydrolysis of starch by amylase.
[3]
(f) State the relationship between pH and the hydrolysis of starch by amylase.
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(g) The student’s hypothesis was:
• the activity of the enzyme would increase with increasing pH.
Discuss the student’s hypothesis in relation to the results obtained.
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[Total : 23]
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Practical 9 – Testing different food stuffs
1. Carry out the iodine in potassium iodide solution test for starch
i.
Carry out the iodine in potassium iodide solution test on a range of different types of starch
(suspensions) to see the range of blue-black colours obtained.
ii.
Test different food substances to identify those containing starch.
2. Carry out the emulsion test for lipids
i.
Use the (ethanol) emulsion test with vegetable oil and yellow-dyed water.
ii.
Use the emulsion test with crushed fruits and seeds.
3. Carry out the biuret test for proteins
i.
Use the biuret test on a solution of egg white, skimmed milk, chicken or tofu and water.
ii.
Carry out a semi-quantitative biuret test. Prepare a set of standard solutions, and then for each add
a fixed volume to the same volume of biuret solution in separate test tubes, mixing the contents
and leaving for a standard length of time. Use the same volumes and time for unknown solutions
and compare the intensity of colour obtained with the standards.
Practical 10 - The identification of biological chemicals present in solutions
This practical focuses on making decisions about measurements and observations, recording and presenting
data and observations, interpretation, drawing conclusions and suggesting improvements. You will also develop
other assessed skills throughout the practical.
Intended learning outcomes
By the end of this practical you should be able to:
• Decide what tests to carry out and what observations to make
• Use an appropriate means to record your observations, constructing any tables before you make the
observations
• Describe and summarise the key points of your observations
• Draw conclusions in terms of the presence or absence of different chemicals in the solutions
• Suggest alternative strategies for identifying some of the materials
Background information
• Make sure that you know how to carry out Benedict’s test, what it is used for and what the positive and
negative results should be.
• Make sure that you know how to carry out biuret test, what it is used for and what the positive and negative
results should be.
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• Think about how Benedict’s test and the enzyme amylase can be used to confirm the presence of a
polysaccharide such as starch.
• Think about how acid hydrolysis, neutralisation and Benedict’s test can be used to confirm the presence of the
non-reducing disaccharide, sucrose.
You will use the materials provided to identify the unknown materials in the solutions
A, B, C and D
• Read and think about the information above.
• The solutions A, B, C, D and E each contain only one of the following materials, but not necessarily in this
order
o
A reducing sugar
o
A non-reducing sugar
o
A polysaccharide that can be hydrolysed by amylase
o
Proteins including amylase
o
No dissolved material
• You are also provided with materials for biuret test and for Benedict’s test, as well as dilute hydrochloric acid,
calcium hydrogen carbonate powder and a waterbath at 35°C
Method
Preparations and making observations
1. You need to decide what tests to do and in what order so that it is possible to use the amylase to test some
of the other solutions.
2. Decide how you are going to record your observations so that it will be absolutely clear what you did to which
solutions, what you observed and your interpretation of the observations.
3. Prepare a piece or pieces of paper in accordance with your decisions.
4. Make a risk assessment of your proposed methods and decide what precautions to take to reduce the
likelihood of an accident and to reduce the damage any accidents might cause – ask your teacher to confirm
that you may go ahead with the tests.
5. Carry out the tests with full regard to safety, recording your observations and interpretations.
6. Record the identity of the unknown solutions.
Write-up
• hand in your original laboratory records, including your methods, observations and interpretations.
• suggest improvements to the method including some of the following:
• a simpler way of testing for the presence of starch,
• starch would also be hydrolysed by acid. Suggest a better order to do your tests if this caused you difficulties,
or a way of using amylase to confirm that it is non-reducing sugar rather than starch that is present,
• if a solution contained a small amount of reducing sugar and also nonreducing sugar, suggest how it might be
possible to use repeated benedict’s tests, filtering the precipitate out after each, to remove the reducing sugar
before testing for non-reducing sugar.
20
Prepared October 2013 by C. Coetzer
Practical 11 – Identify starch, protein and reducing sugar in various solutions
You are provided with five solutions S1, S2, S3, S4 and S5.
These solutions contain starch or protein or reducing sugar in varying concentrations or any two of these or
none of them.
Carry out tests, using only the reagents provided, to identify the contents of each of the solutions. Use your
results to decide whether the concentration of each of the substances you identify is high or low.
You are required to identify two of the solutions, S1 to S5, that could be mixed together to feed a young
mammal. The mixture needs to contain
• a high concentration of starch,
• a high concentration of protein and
• a low concentration of reducing sugar.
The mixture must be made up from equal volumes of only two of the solutions, S1 to S5.
You will need to consider carefully how you will carry out the tests so that you can determine the relative
concentrations of the three substances.
(a) (i) Complete the table below to show how you will carry out the tests on each solution.
Test for
Method for test
[4]
Expected Results
negative
Low concentration
High concentration
Starch
Reducing
sugar
Protein
21
Prepared October 2013 by C. Coetzer
(ii) Prepare the space below and record your observations of the tests on all the solutions.
[6]
(iii) Use your observations to state the two solutions that should be mixed in equal volumes to provide the
correct mixture to feed the young mammal.
.............................................................................................................................. [1]
A student investigated the time taken for the complete digestion of starch by an enzyme found in the saliva of
25 individuals of a species of mammal.
3
A sample of saliva was collected from each individual and mixed with 5 cm of 1% starch suspension. Samples
of the mixture were tested for the presence of starch.
The student recorded the time taken for the complete digestion of starch.
The investigation was repeated with the same individuals on the following day.
The results of the student’s investigation are shown in Table 11.1.
Table 11.1
22
Prepared October 2013 by C. Coetzer
(b) (i) Plot these data shown in Table 11.1.
[4]
(ii) Describe the patterns in the results.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
(iii) Suggest a reason for the difference between the results for day 1 and day 2.
.................................................................................................................................................................................
.............................................................................................................................................................................[1]
(iv) Suggest how you might control the variables in this investigation to compare a different species of mammal
with the mammal studied.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
[Total: 22]
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Prepared October 2013 by C. Coetzer
Practical 12 - Diffusion in liquid
You are provided with a boiling tube containing 10% gelatine.
(a) Melt the gelatine in the tube by immersing it almost up to the rim in a beaker or jar of hot water. While
waiting for it to melt continue with (b).
(b) Use a spirit marker to label three test-tubes A, B and C and make three marks on tubes A and B as shown
in Fig. I, Add your initials to these two tubes.
55mm
mm
Clear gelatine
5mm
Blue gelatine
55mm
Clear gelatine
Fig 1
Fig 2
(c) Pour the liquid gelatine into tubes A and B up to mark 1 and allow it to set. Setting can be hastened by
dipping the tube upright into cold water. Do not allow it to set with the surface oblique.
(d) Pour about 20mm liquid gelatine into tube C and use a dropping pipette to add one drop of methylene blue
solution.* Swirl the tube gently to mix the dye with the gelatin.
Make sure the gelatine in tube A has set and use a clean dropping pipette to transfer enough blue gelatine to fill
the space between marks 1 and 2. Insert the pipette well down inside tube A so that blue gelatine does not
touch the sides above mark 2. Use the pipette to draw off any air bubbles which form.
(e) Add another 9 drops of methylene blue solution to tube C and use the dropping pipette to transfer this blue
gelatine to tube B in the same way as before. Allow the blue gelatine in tubes A and B to set firmly (about 5
minutes in cold water). While waiting, continue with instruction (g).
(f) When the blue gelatin has set, run a little cold water into both tubes to ensure that no liquid gelatin remains.
Pour off the water and fill both tubes up to mark 3 with cool but liquid gelatin and cool it quickly. Cork both tubes
(Fig. 2)
24
Prepared October 2013 by C. Coetzer
(g) In your notebook make diagrams, similar to Fig. I, of tubes A and B to show the position of the gelatin and
the blue dye. Leave space for two more diagrams.
(h) After a week, examine the tubes again and make two more diagrams beside the first two to show the
distribution and intensity of the blue colour. Answer discussion questions 1 and 4 while the tubes are still
available for examination.
* TAKE CARE. Methylene blue temporarily stains the skin and permanently stains clothing.
1. Did the diffusion of methylene blue take place equally upwards and downwards in the tube? Give figures to
support your answer.
.................................................................................................................................................................................
.................................................................................................................................................................................
2. In what direction would you expect diffusion to occur if a drop of methylene blue were surrounded on all
sides by a large volume of gelatine?
.................................................................................................................................................................................
.................................................................................................................................................................................
3. If you did experiment I, comment on the relative speeds of diffusion in air and in gelatine.
................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
4. What difference was there in the rates of diffusion of methylene blue in tubes A and B, bearing in mind that
the concentration in tube B was about ten times greater than in tube A ? Give measurements to support
your answer
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
NOTE. You may not regard gelatine as a true liquid but water would have been unsatisfactory for two main
reasons: (a) it is difficult to start with a distinct boundary between two liquids which can mix and (b) convection
currents can occur and so distribute the dye. The gelatine prevents the water from flowing so that the dye can
move only by diffusion.
Practical 13 – Diffusion in agar blocks (Exam Question)
The blue-green agar blocks contain an indicator.
The investigation involves placing different sizes of agar blocks into dilute hydrochloric acid.
As the acid moves (diffuses) into the agar blocks the indicator loses its blue-green colour.
You are required to investigate the independent variable, surface area to volume ratio of the agar blocks.
You are provided with:
25
Prepared October 2013 by C. Coetzer
You must not touch the agar blocks with your hands. Use the blunt forceps and paper towel to handle the
blocks.
If you get hydrochloric acid on your skin, wash off with cold water.
You are required to prepare four different sizes of agar blocks.
You will require three blocks of each size.
You are advised to read steps 1 to 6 before proceeding.
Proceed as follows:
1. Cut the agar block provided into blocks, each 1 cm × 1 cm × 1 cm. Cut the blocks on the white tile or
chopping board provided.
Fig. 13.1 shows how to obtain blocks with a different surface area compared with the blocks in step 1.
step 1 : 1 cm × 1 cm × 1 cm blocks
Fig 13.1
26
Prepared October 2013 by C. Coetzer
2. Cut two of the 1 cm × 1 cm × 1 cm blocks as shown in Fig. 13.1 to obtain the blocks you need for the next
surface area.
You may use the space on Fig. 1.1 to work out how to cut two further sizes of blocks with different surface
areas.
3. Cut two further sizes of blocks with different surface areas. You will need three blocks of each of the four
sizes of block.
4. Put three of each size of block into the beaker or container using blunt forceps.
5. Put H into this beaker or container to cover the blocks and start timing.
6. Observe the blocks for the disappearance of the blue-green colour. This is the endpoint of the reaction. As
soon as you see the green colour disappear record the time taken for each block to reach the end-point. If any
block remains blue-green at 15 minutes, record “more than 15”.
The different sizes of agar blocks have different surface areas and volumes.
(a) (i) Complete Table 13.1.
[3]
Table 13.1
2
Volume / cm
3
Dimensions /cm
Surface area / cm
Surface area : volume ratio
1x1x1
6
1.0
6:1
1 x 1 x 0.5
4
0.5
8:1
(ii) Prepare the space below and record your results.
[5]
27
Prepared October 2013 by C. Coetzer
(iii) Identify two significant sources of error in your investigation.
................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
................................................................................................................................................................................
.............................................................................................................................................................................[2]
A student investigated the effect of temperature on the rate of breathing in fish. Fish take water in through the
mouth and force it over the gills when the mouth closes. Oxygen diffuses from the water into the gills. The gill
cover opens to allow the water to leave as shown in Fig. 13.2.
Fig 13.2
A fish was placed in a bag partly filled with water. The bag, containing the fish, was placed in a large beaker
containing water at 5 °C.
The number of times the gill cover opened in one minute was recorded. The bag containing the fish was
removed and the temperature of the water in the beaker was raised to 10 °C.
A bag containing a different fish of the same species was then put into the beaker. The number of times the gill
cover opened in one minute was counted.
This procedure was repeated using a different fish of the same species for each temperature as shown in Table
13.2. However, the student decided that the temperature would not be increased to above 25 °C as this might
damage the fish.
(b) (i) Suggest how you might control three of the variables in the student’s investigation.
................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
................................................................................................................................................................................
.................................................................................................................................................................................
.............................................................................................................................................................................[3]
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Prepared October 2013 by C. Coetzer
Table 13.2 shows the results of this investigation.
Table 13.2
(ii) Plot a graph of the data shown in Table 13.2.
(iii) Describe and explain the effect of the temperature on the rate of breathing in this
species of fish.
................................................................................................................................................................................
.................................................................................................................................................................................
................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
[Total: 20]
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Prepared October 2013 by C. Coetzer
Practical 14 - Investigating Osmosis
Method.

You are provided with a solution of sucrose of 1mol dm

You need to have 15 cm of this solution and also 15cm of 0.5 mol dm ,15cm of 0.1mol dm sucrose
3
-3.
3
-3
3
-3
3
and 15 cm of distilled water.

Cut out 13 ‘chips’ using a scalpel and ruler. Each one MUST be exactly the same length (50mm) NB use
mm, not cm. They could all be 48, 49 mm, but they MUST be the same.

Measure and record the length of each chip.

Set up 3 test tubes in a rack. Put 15 cm of each of the solutions into each tube, as in the table below. Write
3
on the tube what solution it contains and mark the level of the liquid in the tube – BEFORE ADDING THE
CHIPS

Place three ‘chips’ into each tube and note the time they go in. Keep the remaining chip in a dry test-tube.

After 30 minutes, remove the chips carefully and mark the new level of the liquid in each tube.

Carefully measure the new lengths of the chips (calculate the average length of the 3 chips for each tube)
and note any difference in the texture of the chips.
Results
Design your own results chart and record the results
Conclusions.
1. Describe how the different concentrations of sucrose were made up.
.................................................................................................................................................................................
.................................................................................................................................................................................
................................................................................................................................................................................
2. Using the information that you have collected explain the effects of each of these 4 solutions on the
chips.
................................................................................................................................................................................
................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
3. Comment on any sources of inaccuracy in this investigation and what you could have done to rectify them.
................................................................................................................................................................................
.................................................................................................................................................................................
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Prepared October 2013 by C. Coetzer
Practical 15 – Effects of different salt solutions on potato chips (Exam Question)
When plant cells are placed into different concentrations of sodium chloride solution, water moves between the
cells and the solutions. This will affect how much a piece of plant tissue can bend.
Fig. 15.1 shows how the angle of bend of a sample of plant tissue can be measured after you have pushed it
until it will bend no further without breaking.
Fig 15.1
You are provided with eight pieces of plant tissue, each soaked in a different concentration of sodium chloride
solution labelled P1, P2, P3 and P4.
P1, P2 and P3 each contain one of the concentrations of sodium chloride solution,
-3
-3
-3
1.00 mol dm , 0.50 mol dm and 0.25 mol dm but not necessarily in that order.
P4 has an unknown concentration of sodium chloride solution.
(a) (i) When samples of the plant tissue from each of the concentrations of sodium chloride solution are put into
water, water will move.
Select the correct words from “least”, “most”, “same” to complete the sentence below.
You may use a word once, more than once or not at all.
The sample of plant tissue soaked in the highest concentration of sodium chloride solution will have the
........................................ bend at the start and when placed in water the bend will change
the........................................ .
[1]
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Prepared October 2013 by C. Coetzer
You are required to:
• Observe and record the effect of putting samples of plant tissue from P1, P2, P3 and P4 into water, W, for
different times up to 10 minutes.
• Use these results to answer (b)(ii) concerning the concentrations of the sodium chloride solutions, P1, P2, P3
and P4.
The size of plant tissue is a variable which needs to be considered. Each piece of plant tissue should be a
standard size.
(ii) Decide how you will make sure that the pieces of plant tissue are standard size.
State the size you will use.
.................................................................................................................................................................................
.............................................................................................................................................................................[1]
(iii) Describe how you will obtain accurate results to record the angle of bend of the plant samples.
............................................................................................................................................................................ [1]
You are advised to read steps 1 to 5 before proceeding.
(iv) To observe the effect of putting samples of plant tissue from P1, P2, P3 and P4 into
W for different times up to 10 minutes you need to decide the times you will use.
State the times at which you will measure the angle of bend.
............................................................................................................................................................................ [1]
Proceed as follows:
1. Cut the pieces of plant tissue as stated in (a)(ii).
2. Measure the angle of bend of the samples of plant tissue from P1, P2, P3 and P4 as shown in Fig. 1.1.
Record your results in (b)(i).
3. Put the samples from P1, P2, P3 and P4 into separate containers and add W to each container so that the
samples are covered.
4. Start timing.
5. Measure the samples at each of the times that you decided in (a)(iv).
(b) (i) Prepare the space below and record your results.
[7]
32
Prepared October 2013 by C. Coetzer
(ii) Use your results in (b)(i) to identify the concentrations of the sodium chloride solutions.
Complete the diagram below to show the position of each of the concentrations P1, P2 and P3. Write “P4”
where it fits in the series of sodium chloride concentrations.
(iii) Explain the effect of putting the plant tissue from P1 into water, W.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
........................................................................................................................................................................... [1]
(iv) Identify two significant errors in this investigation.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [2]
(v) A protractor was used to measure the angle of bend.
State the value of the smallest division on the protractor. ................
State the actual error in using the protractor to measure the angle of bend.
................................................... [1]
(vi) Suggest how you would make this investigation as reliable as possible.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
[Total: 20]
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Prepared October 2013 by C. Coetzer
Practical 16 - Plasmolysis
(a) Use a scalpel or razor blade to take a section of red onion skin.
(b) With a pair of fine forceps lift up a strip of the epidermis at one side of the cut. Lift only the epidermis and
not the underlying cortex. Having freed a narrow band of epidermis, pull it off with the forceps (Fig. 1) and press
it flat on a slide with the outermost surface upwards.
(c) Use the scalpel or razor blade to cut about 10 mm of this strip from the thinnest and reddest portion (Fig. 2)
and, using a dropping pipette, cover this with 3 drops of water.
(d) Use the forceps to lower a cover slip carefully on to the water drops, avoiding trapping air bubbles (Fig. 3),
and examine the epidermis under the microscope using the x10 objective.
(e) Move the slide about to find a group of clearly defined cells near the edge, with red cell sap, and make a
drawing in your notebook to show one of these cells. Draw the cell at least 50 mm long, representing the outline
accurately and shading the area filled with cell sap. Clip the slide securely to the microscope stage and leave it
in this position for the rest of the experiment.
(f) Use the pipette to place 2 drops of sucrose solution on the left-hand side of the slide, just touching the edge
of the cover slip.
(g) Draw all this solution under the cover slip by applying a strip of blotting paper to the right- hand edge of the
cover slip. Try not to move the slide, the cover slip or the epidermis.
(h) Examine the cells again and watch for about 2 minutes. If nothing happens, draw through some more
sucrose solution.
(i) When a significant change has occurred in the cells, draw the same cell as before to show the cell wall and
the cell sap. The cell is plasmolysed.
(j) Use the pipette to place 3 drops of water on the left hand' side of the slide and draw it through under the
cover slip as before. Do this twice to flush out all the sucrose solution.
(k) Study the cells again for about 2 minutes repeating operation (j) if nothing happens in this time.
Draw what you see under the microscope
34
Prepared October 2013 by C. Coetzer
Practical 17 - The effect of temperature on membrane permeability in beetroot
This practical focuses on making measurements and observations, recording and presenting data, analysis,
drawing conclusions and evaluating methods. You will also develop other assessed skills throughout the
practical.
Intended learning outcomes
By the end of this practical you should be able to:
• Experience relevant methods, analysis and conclusion.
• Describe and explain the relationship between temperature and membrane permeability.
• Evaluate procedures
Background information
• The colour of beetroot is due to the presence of a red pigment called anthocyanin
• The cell membrane is mainly made up of two types of molecules, phospholipids and proteins scattered around
in the membrane.
• The membrane is partially permeable
• Protein structure is denatured at high temperatures
You will investigate the effect of temperature on the permeability of the cell membrane in beetroot.
• Read the information above
• Identify and write down the dependent and independent variables
• Write down a hypothesis
• Draw a sketch graph to show what you think will happen
• Identify any variables that should be controlled and outline how this should be done
A colorimeter should be used to compare the colours of anthocyanin solutions obtained.
Method
Preparations and making observations
1. Use a cork borer to cut cylinders of fresh beetroot tissue. Place on a tile and cut into 30 discs, each 3mm
wide.
2. Place all the discs in a small beaker and wash under a running tap for at least five minutes.
3. Label six test tubes – 30°C, 40°C, 50°C, 60°C, 70°C, 80°C.
4. Add 10cm3 cold distilled / de-ionised water to each tube.
5. Set up a water bath using a large beaker, tripod, gauze and Bunsen burner.
6. Heat the water gently until a temperature of 80oc is reached then remove heat source.
7. Take five of the beetroot discs and impale on a mounted needle with space between each disc.
8. Immerse the discs in the water bath for exactly one minute, then remove and carefully push the discs into the
test tube labelled 80°C and set aside.
9. Reduce the temperature of the water bath to 70°C and take a second set of five discs and repeat the process
of immersion for one minute followed by putting them into the next tube.
10. Continue the process for each of the temperatures.
11. After the discs have stood for thirty minutes shake the tubes and pour this liquid into a cuvette.
12. Fill a second cuvette with distilled water.
35
Prepared October 2013 by C. Coetzer
13. Place a blue filter into the colorimeter and use the distilled water to zero the machine adjusting the pointer
to zero absorbance.
14. Measure the colour density of the 70°C solution.
15. Wash out the cuvette and repeat the procedure to record the light absorbance for each of the temperatures.
Write-up
• Record your results in a clear table ensuring units are put in headers where possible.
• Plot a graph of relative concentration of pigment against temperature.
• Explain your findings using your knowledge of cell membranes.
• Assess the reliability of the results obtained and suggest any modifications you could make to improve the
experiment
Discussion / evaluation points should include:
• explanation of the shape of the graph
• consistency of the pigment within the beetroot
• why the discs were washed before heating
• possible problems with the method e.g. impaling onto a mounted needle
• for students unable to obtain a full set of results the following could be used for analysis
36
Prepared October 2013 by C. Coetzer
Practical 18 - Investigating the action of the enzyme Catalase

All living things make hydrogen peroxide as a waste product of cell reactions.

Hydrogen peroxide is harmful to living things so must be broken down immediately.

The enzyme called catalase speeds up this reaction.
2H2O2 ----------- 2H2O + O2
Your source of catalase is potato slices. The catalase is distributed throughout the slice but it is only the surface
catalase that catalyses the reaction.
You are going to investigate the effect of the amount of enzyme has on the reaction rate.
Method and apparatus.
1. Cut 25 slices of potato (about 2-3mm thick) from a chip 15mm square.
3
2. Set up a test-tube in a rack and pour into it 15cm of H2O2.
3. Put 1 square of potato in the test-tube then connect the delivery tube into the test-tube with water.
4. Count the number of bubbles of gas that emerge from the delivery tube in 1 minute and record this number
5. Repeat for one more minute and another.
6. Repeat this for the other number of slices using fresh slices and hydrogen peroxide.
Results
Number of slices
st
1 minute
nd
2 minute
rd
3 minute
Average per
minute
1
2
4
8
10
37
Prepared October 2013 by C. Coetzer
Plot a line graph of number of slices (x-axis) versus average number of bubbles
(5)
Conclusions.
1.
Describe the relationship of the effect of the number of slices of potato on the number of bubbles
produced.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.............................................................................................................................................................................(2)
2. Explain why it was necessary to use fresh hydrogen peroxide for each time you used different slices. Use
the terms substrate, enzyme and products.
(2)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
3. List 2 inaccuracies in the method of this investigation and how they might have affected the results
(4)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
38
Prepared October 2013 by C. Coetzer
.................................................................................................................................................................................
.................................................................................................................................................................................
4. Imagine that you collected results for the 10 slices and your results were 110, 90, 70 bubbles per minute.
Would you regard these results as valid? Explain your answer.
(3)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
Practical 19 - Investigating the effect of substrate concentration on the rate of an
enzyme controlled reaction.
Saliva contains the enzyme salivary amylase that hydrolyses cooked starch to maltose. You are provided with a
1% suspension of cooked starch, usual laboratory apparatus, iodine solution, clingwrap. You are to provide
3
your own 10 cm sample of saliva!
You are to investigate the effect of varying the substrate concentration on the rate of hydrolysis of starch.
An appropriate number of concentrations (treatments), in the time available, would be a minimum of 5. Each
treatment should be replicated, but in this specific practical that may not be possible.
Method.

Put a piece of Clingwrap on a sheet of folio paper on the desk. Place spots of iodine solution 1 cm apart
in the rows and columns.

3
Label 5 test-tubes as follows: 1%, 0,75%, 0,5%, 0,2%, 0,05%starch and place 5cm of each
concentration into the appropriate test-tube.

Set up a water-bath ( a 400cm beaker approximately half full with water at 35°C )

Put 2cm of saliva into each of 5 test-tubes and place these into the water-bath.

Pour one of the samples of saliva into the 1% starch tube and shake to mix. Note the time and
3
3
immediately add 2 drops of this mixture on to the first drop of iodine solution. After 2 minutes add 2 more
drops of the starch / saliva solution to the next drop of iodine solution, repeat at 2 minute intervals until
you reach what you decide to be the ‘end-point’ (ie when starch has been hydrolysed), record the time
taken to reach this point.

Repeat this procedure with the other starch concentrations.
39
Prepared October 2013 by C. Coetzer
Results
Starch concentration
Time /min to reach the
Rate of reaction
(%)
end point
1 / time
Plot a graph of these results. ( Rate of reaction is 1/time to reach end point.)
(6)
Conclusions
1. Using these results and your own knowledge of enzyme-mediated reactions, describe and explain the
effect of the substrate concentration on the rate of this reaction.
(6)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
40
Prepared October 2013 by C. Coetzer
.................................................................................................................................................................................
.................................................................................................................................................................................
2. Describe how you could determine that this reaction is controlled by an enzyme.
(3)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
3. Describe the evidence that you have seen that suggests that there are intermediate products formed during
this reaction.
(2)
.................................................................................................................................................................................
.................................................................................................................................................................................
4. Describe how you could determine that full hydrolysis had occurred and how you could determine the
identity of the end products.
(5)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
5. List 2 points on inaccuracy in this investigation and how these may have affected your results.
(4)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
6. List and explain 2 ways of improving the reliability of your results.
(4)
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
Practical 20 – Yeast and its enzymes (Exam question)
Yeast cells contain enzymes, which catalyse the breakdown of glucose to produce ethanol and carbon dioxide.
These products change the environment of the yeast cells and can affect their activity and survival.
The carbon dioxide when dissolved forms a weak acid so the more carbon dioxide that is released the more
acid will be formed.
You are required to investigate the effect of different concentrations of ethanol on the activity of yeast cells by
measuring the change in pH, using Universal Indicator paper.
41
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You are provided with:
• five labelled tubes each containing 0.7 g of dried yeast
• at least 50 cm3 ethanol, labelled E
• at least 100 cm3 20% glucose solution, labelled G
• at least 100 cm3 distilled water, labelled W
Ethanol is harmful and highly flammable. If any comes into contact with your skin, wash immediately
under cold water.
It is recommended that you should wear eye protection.
Keep the ethanol covered when you are not using it.
It is important to find the pH of the glucose solution and the ethanol before starting the investigation.
Place a small piece of the Universal Indicator paper on a white tile.
Using a clean pipette, place a drop of glucose solution onto the paper.
Use the colour chart to identify the pH.
Repeat, after cleaning the pipette, to find the pH of ethanol.
(a) (i) Record the colour of the Universal Indicator paper and the pH for the glucose solution and the ethanol
below.
[2]
Clean and dry the tile.
You are going to change the independent variable, the concentration of ethanol.
Table 20.1 shows how to make up two of the concentrations you should use.
Table 20.1
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Prepared October 2013 by C. Coetzer
(ii) Using the information in Table 20.1, decide which other concentrations to make and complete Table 20.2
including the concentrations from Table 20.1.
Table 20.2
Tubes 1 to 5 each contain the same mass (0.7 g) of dried yeast.
1. Adding the water before the ethanol, use the syringes provided to make up the ethanol concentrations in
the correct tubes.
2. Use the beaker, or other container provided, to make a water bath with warm water between 45 °C and 50
°C.
3. Shake the tubes carefully to thoroughly mix the ethanol and water.
4. Place the tubes into the warm water and leave them for at least 5 minutes.
5. Use the marker pen provided to label the white tile as shown in Fig. 1.1.
6. Arrange two rows of small pieces of Universal Indicator paper on the white tile as shown in Fig. 20.2.
Fig 20.2
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Prepared October 2013 by C. Coetzer
7. After the tubes have been in the water bath for at least five minutes start a stopwatch or stop clock or note
the time on a clock.
8. Use a clean 10 cm3 syringe to put 10 cm3 of the glucose solution into each tube starting with tube 1.
Each time shake the tube well and return it to the warm water bath.
9. When the clock shows one minute, use the glass rod to remove a drop from the contents of tube 1 and place
the drop onto the correct piece of Universal Indicator paper.
10. Clean the glass rod and use it to remove a drop as described in step 9 from the other four tubes.
11. When the clock shows 10 minutes, use the glass rod to take further drops as described in step 9.
(iii) Prepare the space below, to record both the colour of each piece of Universal Indicator paper and the pH.
[4]
(b) (i) Identify a significant source of error in this investigation.
.............................................................................................................................................................................
......................................................................................................................................................................... [1]
(ii) You used syringes to measure the volumes of ethanol and water.
State the volume of the smallest division on the syringe ………………
State the degree of uncertainty ………………………………
[1]
A student decided to investigate the effect of temperature on the activity of enzymes in yeast.
The student measured the activity of the enzymes by counting the number of bubbles of carbon dioxide, which
were released in three minutes.
The results of the student’s investigation are shown in Table 20.3.
Table 20.3
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(c) (i) Plot a graph of the data shown in Table 20.3.
(ii) From the graph, estimate the enzyme activity at 25 °C.
..................................................................................................................................................................... [1]
(iii) Suggest how the student should make sure that the results of this investigation are as accurate as possible,
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
as reliable as possible.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
In carrying out this investigation the student made the hypothesis that:
The activity of the enzymes in yeast increases as temperature increases.
(d) State whether you think that this hypothesis is supported by the student’s results.
Explain your answer.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [2]
[Total: 21]
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Prepared October 2013 by C. Coetzer
Practical 21 – Enzyme hydrolysis of starch to glucose (Exam question)
Enzyme E catalyses the hydrolysis of starch to glucose.
The end-point of the reaction can be found by measuring the time taken for all the starch to be hydrolysed.
You are required to investigate the effect of the independent variable, copper sulphate concentration, on
enzyme E.
You are provided with:
Copper sulfate can inhibit enzyme E.
The extent of inhibition depends on the concentration of the copper sulfate solution. A student investigated the
inhibition of enzyme E at concentrations of copper sulfate solution greater than 0.03% and found that the
enzyme was completely inhibited.
The student suggested the hypothesis:
concentrations of copper sulfate solution below 0.03% will continue to inhibit the enzyme.
You are required to investigate this hypothesis by carrying out a serial dilution of copper sulfate solution which
reduces the concentration by ten-fold between each successive dilution.
Fig. 21.1 shows how to make the first concentration of 0.003% copper sulfate solution.
Fig 21.1
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Prepared October 2013 by C. Coetzer
(a) (i) Complete Fig. 21.1 to show how you will make two further concentrations of copper sulfate solution.
[3]
Proceed as follows:
1. Prepare the concentrations of copper sulfate solution as shown in Fig. 1.1 in the containers provided. Use
the syringe labelled ‘For copper sulfate’.
2. Label test-tubes with the concentrations of copper sulfate solutions and label another test-tube W.
3. Wipe the tile clean with a damp paper towel and then dry the tile. Label the tile, as shown in Fig. 21.2. The
numbers indicate the sampling times in seconds.
Fig 21.2
4. Put one drop of iodine on the tile at each sampling time, as shown in Fig. 21.2.
3
5. Put 1 cm of W into the labelled test-tube.
3
6. Put 3 cm of S into the same test-tube. Mix well.
3
7. Put 0.5 cm of E into the same test-tube. Mix and start timing.
8. Use a glass rod to stir the mixture.
9. After 15 seconds use the glass rod to transfer a drop of the mixture to the iodine drop, labelled 15, on the
tile.
10. Immediately clean the glass rod with a paper towel.
11. Repeat steps 8 to 10 at 15 second intervals until the iodine drop does not change colour. If the iodine drop
changes colour at 180 seconds, record ‘more than 180’ as your result (for step 12).
12. Record the time taken to reach the end-point.
13. Repeat steps 3 to 12 replacing the 1 cm3 of W with 1 cm3 of the lowest concentration of copper sulfate
solution.
14. Repeat step 13 with the other concentrations of copper sulfate solution.
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Prepared October 2013 by C. Coetzer
(ii) Prepare the space below and record your results.
[5]
(iii) The student’s hypothesis stated that “concentrations of copper sulfate solution below 0.03% will continue to
inhibit the enzyme”.
Explain how your results provide evidence for the support or the rejection of this hypothesis.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [2]
(iv) Identify one significant source of error in your investigation.
.................................................................................................................................................................................
.............................................................................................................................................................................[1]
(v) A colorimeter could have been used to determine the end-point.
Describe three other modifications to this investigation which would improve the confidence in your results.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................ [3]
Table 21.1 shows the results of an investigation into the effect of the concentration of copper sulfate solution on
a protein suspension. A protein suspension was mixed with different concentrations of copper sulfate solution.
After a set time, the percentage absorbance of light was measured using a colorimeter.
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Prepared October 2013 by C. Coetzer
Table 21.1
(b) (i) Draw a circle around each of the anomalous results and complete the table.
[2]
(ii) Plot a graph of the data shown in Table 21.1.
(iii) Explain the effect of copper sulfate solution on the protein suspension.
.................................................................................................................................................................................
.................................................................................................................................................................................
.................................................................................................................................................................................
............................................................................................................................................................................[2]
[Total: 22]
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Prepared October 2013 by C. Coetzer
Practical 22 - The effect of wind speed on the rate of transpiration in a leafy shoot
This practical focuses on making measurements and observations, recording and presenting data, analysis,
drawing conclusions and evaluating methods. You will also develop other assessed skills throughout the
practical.
Intended learning outcomes
By the end of this practical you should be able to:
• Identify dependent and independent variables
• Make a hypothesis and express this in words and graphically
• Identify the variables that should be controlled
• Experience relevant methods, analysis and conclusion.
• Describe and explain the relationship between transpiration and wind speed
• Evaluate procedures
Background information
• Transpiration is the movement of water through plants, from the roots where it is absorbed by osmosis, to the
leaves where it is lost by evaporation.
• Water leaves a plant’s leaves through stomata, the aperture of which is controlled by guard cells.
• Most plants open their stomata during the day and close them at night.
• Plants may close their stomata when stressed by losing too much water.
• The real purpose of stomata is to absorb carbon dioxide and release oxygen from photosynthesis without
losing too much water.
• The evaporation of water is affected by wind speed, temperature, humidity and atmospheric pressure.
You will investigate how wind speed affects the rate of transpiration from a leafy shoot by using a potometer.
• Read the information above
• Identify and write down the dependent and independent variables
• Write down a hypothesis
• Draw a sketch graph to show what you think will happen
• Identify any variables that should be controlled and outline how this should be done
• What would be the best method for setting up the potometer?
• Plot a graph of the distance of the fan from the shoot, against the rate of water movement in the potometer,
after the practical. Make sure you know how to calculate rate.
Wind speed in this case is varied by moving the fan to fixed distances from the leafy shoot. Although the wind
speed from the fan will not accurately follow the inverse - square law, you would be well advised to understand
how increasing the distance of the fan from the shoot, may affect the wind speed.
Method
Preparations
The apparatus should be assembled as shown in the following diagram.
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Prepared October 2013 by C. Coetzer
1. Attach the rubber tubing, the capillary tube and the water reservoir to the T piece.
2. Fill the reservoir, capillary tube and rubber tubing with water. This can be done by placing them under water
and gently squeezing the rubber tubing until all the air has been removed.
3. Leave the apparatus under water.
4. Cut a fresh leafy shoot with a sharp knife and immediately place the cut end under water.
5. Carefully attach the cut end of the shoot to the rubber tubing. This should be done with the cut end only
under water.
6. Close the tap on the water reservoir.
7. Remove the apparatus from the water and attached to a clamp stand or support.
8. Place a mm scale behind the capillary tube.
9. Place a fan at a set distance from the leafy shoot. Do NOT switch on.
Making observations
1. Note the position of the air bubble in the capillary tube. It may be very close to then end of the tube.
2. Record the time taken for the air bubble to move a set distance along thetube. You will have to determine
this distance base on the speed of thebubble. If the bubble is moving quickly the distance will need to be larger
than if it is moving slowly.
3. Reset the air bubble to then end of the capillary tube by carefully openingthe tap on the water reservoir.
4. Turn on the fan and repeat the procedure.
5. Reset the apparatus and move the fan to another distance.
6. Repeat the procedure with the fan at at least five differetn distances.
You are advised to start with the fan at the furthest distance and gradually move it towards the leafy shoot.
Calculations
1. Calculate the rate of movement using 1/time taken for the air bubble to travel a set distance.
2. Record the rate of travel for each distance in the class result table on the board or flip chart.
3. When all of the results have been recorded in the class results table, calculate the mean rate of movement
for each distance.
4. (Optional) – calculate the standard error for each distance.
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Write up
• Record your results in a clear table ensuring units are put in headers.
• Plot a graph to show the mean rate of movement for each distance.
• (Optional – add error bars to your graph)
• Make an evaluation considering:
• the limitations of the method used,
• anomalous values if any,
• replication and range of values of independent variable,
• the confidence with which the conclusions should be drawn.
• Draw conclusions considering:
• detailed description of the features of the results,
• whether your results agree or contradict your hypothesis,
• a scientific explanation of your results and conclusions,
• any modifications you could make to improve the experiment.
Useful information
• The evaporation of water from a leaf is affected by wind speed, temperature, humidity and air pressure, the
first three having the most significant affect.
• Increased temperature increases the kinetic energy of the water molecules thus increasing the rate of
evaporation.
• Increased wind speed blows away evaporated molecules form around the opening of the stomata thus
maintaining a greater diffusion gradient for the water molecules. • Increased humidity lowers the concentration
gradient and thus slows down the rate of evaporation.
• Reduced air pressure increases the rate of evaporation.
• Factors such as light are affected by the inverse square law where doubling the distance reduces the light
intensity by a factor of four. Although this does not hold true for wind speed, you should be aware that doubling
the distance of the fan from the leafy shoot, will not necessarily mean that the wind speed is reduced by half.
• Possible variables to control include temperature and humidity.
For students unable to obtain accurate data, the following table of results may be used.
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Practical 23 - Potometer Practical

Four potometers were set up in different environmental conditions during daylight.

The length of the water column was measured at regular intervals.

The results are shown in the table below.

‘Still’ means the air is not moving.
Environmental
condition
Length of water column /mm
0 min
30 min
60 min
90 min
120 min
150 min
O
150
150
150
150
150
150
O
150
130
120
95
75
50
O
150
125
110
90
45
15
O
150
140
125
missing
100
90
10 , still, dry
25 , still, dry
25 windy, dry
10 ,still, humid
Plot four lines of these results on ONE set of axes, time on the x-axis, length of water column on the y-axis.
(6)
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Prepared October 2013 by C. Coetzer
Conclusions
1. Which set of conditions gave the fastest water uptake?
(1)
......................................................................................................................................................................
2. Calculate the average rate of water uptake during this experiment, for the plant in question 1. This can be
calculated by dividing the volume of water taken over the entire period by the time taken, in minutes. Show
your working out.
3. Explain why these conditions gave the fastest rate.
(3)
(3)
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
4. Use your graph to determine the likely value of the ‘missing’ data.
Indicate on the graph how you determined this figure.
................................................................................................................................................................... (2)
5. List THREE factors that should be kept the same to make this a fair test.
(3)
.................................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
Explain why these figures measure the rate of water uptake and not transpiration rate.
(2)
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
.............................................................................................................................................................................
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Prepared October 2013 by C. Coetzer
Practical 24 - Stomatal Density in Agapanthus
1. (a) You are required to investigate the stomatal density and an average number of stomata on the lower
leaf surface of Agapanthus. This information can be compared to the upper leaf surface.

You are provided with graph paper where you can calculate the area of one square.

Draw around the leaf and count the number of squares covered by the leaf.

Calculate the surface area of the lower leaf blade. (show your working below)
(b) Calculate the field of view under a low magnification using the eyepiece graticule where the area of a
2
circle is πr . (Show your calculation below)
(c) i) Prepare the space below to record the results of stomatal density of the lower surface of
Agapanthus
ii) Prepare a section of Agapanthus

Bend the leaf over your finger and use forceps to pull a section of epidermis.

Drop immediately into water.

Mount a section of the epidermis onto a slide and cover with a cover slip.

Examine under the low power magnification and count all the stomata in the field of view.
(d) Calculate the number of stomata on the whole leaf.
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Practical 25- Pathways for gases in a leaf
(a) Paint an area about 10 cm square on both the upper and lower surface of a leaf with a thin layer of clear
nail varnish and leave the varnish to dry for 10 minutes. Meanwhile, continue with instruction (b).
(b) Fill a beaker two thirds full with water (hot water if available) and heat it on a tripod and gauze over a
Bunsen burner until it reaches about 70 °C.
(c) Extinguish the burner when the water is hot enough.
(d) Hold a leaf (not the one with the nail varnish) in forceps and plunge it into the hot water (Fig. 25.1), whilst
observing the lower surface of the leaf.
(e) Repeat the experiment with a fresh leaf but this time watch the upper surface.
(f) If you followed instruction (a), use fine forceps to peel the dried varnish from the lower surface, place it on a
slide and examine it under the microscope. Only a small piece of peel is needed.
(g) Count the number of stomata visible in the field of the microscope and record the results in your notebook. If
the stomata are too numerous to count over the whole field of vision, count only those in, say, a quarter of the
field or between marks on the slide. Alternatively, use a higher magnification if available. (See Fig. 2 on p. 9.02)
(h) Repeat the operation with the nail varnish from the upper surface.
Discussion
1 What did you observe when the leaf was placed in hot water
while watching
(a) the lower surface, .....................................................................
..........................................................................................................
(b) the upper surface? ....................................................................
..........................................................................................................
2 What was the function of the hot water in this experiment?
..........................................................................................................
3 Judging from your varnish peels, which surface of the leaf had
the greater number of stomata?......................................................
..........................................................................................................
Fig. 25.1
4 Use your results and your knowledge of leaf anatomy to explain
your observations in this experiment...............................................
..........................................................................................................
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Prepared October 2013 by C. Coetzer
Practical 26 - Vital capacity and tidal volume
Vital capacity
(a) Put water in the bowl to a depth of about 50 mm. Fill the container with water from the tap and put on the lid
or stopper.
(b) Hold the container upside down in the bowl, with the lid or stopper under the water. Remove the stopper
under water without letting air in. Some water will escape if the container is made of plastic but this does not
matter.
(c) Push a length of rubber tubing through the mouth of the container to position A (See Figure 26.1). Take a
deep breath and blow through the tube until you can exhale no more.
(d) All the air you have breathed out will collect in the container and by comparing the water level inside with
the graduations marked on the container you will obtain an approximate measure of the maximum volume of air
which your lungs can breathe in and out. This is called your vital capacity.
(e) If you intend to repeat the experiment, you must remove enough water from the basin to restore the level to
the 50 mm mark.
Tidal volume
(f) When you have measured your vital capacity, leave the air in the container and the bowl nearly full of water
but push the rubber tubing further through the neck of the container until it is above the water level inside,
position B (See Fig. 26.1).
(g) Blow through the tube just enough to clear out any water trapped in it and raise or lower the container until
the water level inside and outside is the same and at a convenient mark, e.g. the 4 or 3.5 litre mark.
(h) Place the end of the rubber tubing in your mouth and breathe in and out a few times as normally as
possible, holding the container and allowing it to move up and down in the bowl so that the water levels inside
and outside remain the same.
(i) By watching the graduations on the side, you will be able to see approximately how much air you exchange
when you breathe normally. This is called the tidal volume.
(j) If a second person is to use the apparatus, the rubber tubing should be rinsed in disinfectant and washed
under the tap.
Fig 26.1
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Discussion
1 What results did you get for (a) vital capacity,(b) tidal volume?
2 Why do you think the vital capacity is so much greater than the tidal volume, i.e. if you need to exchange only
a few hundred cubic centimetres of air in breathing, what is the advantage of having a lung capacity of several
litres? ..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
..............................................................................................................................................................................
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Prepared October 2013 by C. Coetzer