CORE PRACTICAL ONE

Describe how the effect of caffeine on heart rate in Daphnia can be investigated practically, and
discuss whether there are ethical issues in the use of invertebrates.

Daphnia, the water flea, is a small freshwater crustacean which lacks physiological methods of
maintaining a constant body temperature. This means that if the environmental temperature changes, its
body temperature does so too and its metabolic rate will be expected to rise or fall accordingly. So the
temperature of the organism must be kept constant during the procedure.
In this investigation we shall test the hypothesis that as the concentration of caffeine changes, the
heartbeat rate (cardiac frequency) of Daphnia also changes. Fortunately Daphnia is relatively
transparent and its heart can be seen quite easily under the low Power of the microscope.

Setting up the experiment

1. Select a large specimen and, with a pipette, transfer it to the centre of a small, dry Petri dish. With filter
paper remove excess water from around the specimen so that it is completely stranded.
2. With a seeker place a small blob of silicone grease onto the floor of the Petri dish. Then wipe the
needle clean and use it to gently push the posterior end of the animal into the grease so that it is firmly
anchored. Now fill the Petri dish with water at 300C.
3. Place the Petri dish on the stage of a microscope and observe the animal under low Power. The figure
above shows the position of the heart, watch it beating. Don't confuse the beating of the heart with the
flapping of the legs.
4. Surround the animal with a circular heating coil and fix it in position as shown in the figure below. Also
clamp a small mercury thermometer, or the temperature probe of a digital thermometer, into position.

Estimating the cardiac frequency

 A convenient way of doing this is to time how long it takes for the heart to beat 50 times. If it is beating
too frequently for every beat to be counted, make a mark on a piece of paper every tenth beat. Do
several practice runs to get used to the technique when you feel ready, proceed as follows:
Replace the distilled water in the Petri dish with caffeine solutions of concentration 1mol dm-3 at 30°C.
Estimate the cardiac frequency.
 Switch on the heater so that the water gradually warms up. If the temperature of the water rises too
rapidly, switch off the heater and, if necessary, add a few ice chippings. Estimate the cardiac
frequency at caffeine concentrations of 2 mol dm-3, 3 mol dm-3, 4 mol dm-3, 5 mol dm-3 and 6 mol dm-3,
noting the temperature each time.
Compiled by Stafford Valentine Redden 1
 Present your results in a table and. if you have sufficient readings, draw a graph of the cardiac
frequency as a function of the caffeine concentration.
Ethical issues in the use of invertebrates
Animals are often used for research to enhance scientific knowledge. Monkeys are commonly used for
brain research, dogs are used in behavioural experiments, rabbits and mice are often dissected in
laboratories, mice and fruit flies are used in genetic research, etc. Is it cruel and unfair to utilise these
organisms for research? The issue is very controversial and the ethical guidelines vary from country to
country and person to person. In the UK it is considered ethical to use invertebrates, such as Daphnia in
scientific studies, for the following reasons: Daphnia has reduced awareness of pain because of the lack
of a well developed nervous system. It is transparent and its heart is visible without the need for
dissection. Daphnia is abundant in nature and there is no threat to it or its dependent species (food
chains). Some people also feel that it is bred for fish food and will thus die anyway. Daphnia can
reproduce asexually and may be clones, therefore there is no loss of genetic variation.

Factors to be controlled
- Size of daphnia
- Habitat from which daphnia is obtained
- Temperature of the surrounding
- Oxygen concentration of the water surrounding the daphnia

If daphnia is treated with a chemical,

- The volume and concentration of the chemical should be controlled
- The duration of exposure to the chemical should be controlled
- Time should be allowed for acclimatisation

SAQ1. Sam studied the effect of varying the concentration of the stimulant drug caffeine on heart rate.
She chose to use the water flea, Daphnia, for ethical reasons. In her study, Sam attempted to keep the
temperature of the various caffeine solutions constant. As an extension of this work, she decided to
investigate the effect of temperature on heart rate in more detail. In this new investigation Sam used a
small glass chamber which could hold the Daphnia and water at a set temperature. The whole apparatus
could be placed under a microscope so that the Daphnia heart could be seen. She videoed four Daphnia
at each of five different temperatures for 30 seconds. She used a slow motion replay of the video to count
the number of heart beats in 30 seconds for each Daphnia at each temperature. Her data are summarised
in the table below:

(a) (i) State and explain one ethical reason why Sam chose to use Daphnia for this investigation.
(ii) Suggest one reason for her choice of maximum temperature (30 °C) and one reason for her choice of
minimum temperature (5 °C) used. (2)
(iii) In her investigation, how did Sam try to ensure the reliability of her data? (1)
(iv) Which aspect of her investigation was improved when Sam decided to video the Daphnia? (1)
(b) (i) Calculate the mean heart rate in beats per minute for each temperature. Write your answers in the
spaces provided in the table. Show your working in the space below. (3)

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(ii) Use these data to plot a fully-labelled graph to show the effect of temperature on the mean heart rate of
Daphnia. On your graph, show the variability of the data.

(c) In order to get some idea of the validity of her data, Sam searched the Internet for similar studies. She
could not find any studies that had used her method exactly, especially the video technique, but she did
find data from studies in which direct observation had been used to count heart rates in Daphnia. She
compared the results from one such study, shown in the following table, with her own.

(i) State one similarity and one difference in the conclusions Sam could make about the effect of
temperature on Daphnia heart rate, based on these two sets of data. (2)
(ii) Suggest one explanation for the similarity and one explanation for the difference you have given
above. (4) SNAB SAM 2008

SAQ2. Daphnia (water fleas) can be used to determine the effect of chemicals on heart rate.

(a) (i) Explain one reason why Daphnia is a suitable organism for this experiment. (1)
(ii) An experiment was carried out to investigate the effect of caffeine on the heart rate of Daphnia. State
two variables that you would need to control to produce reliable results. (2)
(iii) Suggest why Daphnia needs a heart and circulatory system. (2)
(b) Caffeine increases human heart rate. Suggest and explain why high caffeine consumption could
increase a person’s risk of developing cardiovascular disease. (2) SNAB Unit 1 Jun 2005

SAQ3. (a) The photograph below shows Daphnia (a water flea). It is a small animal that lives in freshwater
Compiled by Stafford Valentine Redden 3
Daphnia has a heart which pumps fluid around its body. This fluid has a higher solute concentration than
the freshwater that Daphnia lives in.
The table below gives four statements concerning transport in Daphnia. If a statement is correct, place a
tick (J) in the box to the right of that statement and if a statement is incorrect, place a cross ( x ) in the box.
Statements about transport in Daphnia Tick or cross

(i) The movement of fluid through the heart is an example of mass transport

(ii) Daphnia uses diffusion to transport oxygen into muscle cells

(iii) Daphnia tends to lose water to the freshwater by osmosis

(iv) Daphnia can use active transport to move ions from the freshwater into its body

(b) A student investigated the effect of caffeine on the heart rate of Daphnia. Three different Daphnia were
used, A, B and C. The table below shows her results at the end of the investigation.
Daphnia Caffeine Duration of Number of
concentration/ observation
/ seconds heart beats counted
arbitrary units

A 5 10 50

A 5 10 53

A 5 10 47

B 10 10 73

B 10 10 76

B 10 10 76

C 15 10 101

C 15 10 99

C 15 10 100

(i) Calculate the mean number of heart beats per 10 seconds for each Daphnia
Daphnia A .............................. heart beats per 10 seconds
Daphnia B .............................. heart beats per 10 seconds
Daphnia C .............................. heart beats per 10 seconds

(ii) Use your answers from (i) above to predict the mean number of heart beats in 10 seconds for another
Daphnia placed in a caffeine concentration of 35 arbitrary units. (1)
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(iii) Suggest three reasons why the prediction you made for (ii) above may not be very reliable. (3)
(Total 9 marks) SNAB Unit 1 Jan 2008

SAQ4. (a) Daphnia heart rate increases when Daphnia are given the stimulant, caffeine. A student used
this knowledge to estimate the caffeine content of three drinks. To do this she set up a calibration curve.
Initially, she placed one Daphnia in pond water with no caffeine and counted the number of its heart beats
in one minute. She found this to be 180. She then placed the Daphnia in pond water with 10 mg of
caffeine per 100 cm3 of pond water and recorded the time taken for 180 heart beats. This was repeated
for several different caffeine concentrations and the results are shown in the calibration curve below. All
measurements were taken at 15 0C.

The student now repeated the study, using the same Daphnia and keeping the temperature at 15 0C
throughout, but with instant coffee as the source of caffeine. She recorded the time taken for 180 heart
beats to occur. She then repeated this using the two other drinks. The results are shown in the table
below. Use the calibration curve to complete the third column of the table.

(b) By using the same Daphnia throughout the investigation, the student was able to control certain
variables that could have affected her results. Give three variables that the student controlled by using the
same Daphnia. (3)
(c) At the end of the investigation the student removed the Daphnia from the tea and placed it in pond
water. She then recorded its heart rate and found it to be 190 beats per minute. Suggest two reasons why
the Daphnia heart rate was higher at the end of the investigation compared to the 180 beats per minute at
the start. (2) (Total 8 marks) SNAB Unit one

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 CORE PRACTICAL TWO

Describe how to investigate the vitamin C content of food and drink.

PROCEDURE
Add Vitamin C solution of a known concentration (CONCA), drop by drop, with a pipette, to 2 cm3 of the
DCPIP (blue) solution in a test tube. Shake the tube gently after the addition of each drop and continue to
add drops until the DCPIP solution is decolourised. Record the exact volume of vitamin C (VOLA) you
added. Repeat the procedure and calculate the mean volume.

Repeat the procedure with the fruit juice, containing vitamin C at unknown concentration (CONCB).
Record the volume of juice (VOLB) required to decolourise 2 cm3 of the same concentration of DCPIP
solution.
Note: If only one or two drops of fruit juice are required to decolourise DCPIP, dilute the juice five times
and try again.
Using the same technique, compare the vitamin C contents of different food and drinks.
Use the equation below to estimate the concentration of vitamin C in the fruit juices.

CONCB = (VOLA X CONCA) / VOLB

VOLA = Volume of vitamin C solution in ml

CONCA = Concentration of vitamin C solution in mg ml-1
VOLB = Volume of fruit juice in ml
CONCB = Concentration of vitamin C in fruit juice in mg ml-1

SAQ5 Vitamin C is a water-soluble vitamin which is found in foods of plant origin. It is unstable and
easily destroyed.
An investigation was carried out into the effects of boiling on the vitamin C content of some
vegetables. The vitamin C content of each of the vegetables was determined in their raw state and
after boiling.
The results are shown in the table below.

(a) Complete the table below to make a reliable comparison of the effect of boiling on the vitamin C
content on each vegetable. (2)
(b)(i) Describe how the vitamin C content of the fresh vegetables could be determined. (4)
(ii) State three precautions which would need to be taken when carrying out the investigation your
described in (b) (i) (3)
(c) Suggest two ways, other than boiling, in which vitamin C may be lost from fresh fruit and
vegetables. (2) [Total 11 marks]

SAQ6. Give an account of an experiment you could carry out to investigate the effect of storage time
on the ascorbic acid (vitamin C) content of fresh orange juice. [Total 10 marks]

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SAQ 7 Some students carried out an investigation into the effect or temperature on the vitamin C content
of orange juice. They extracted 600 cm3 of orange juice from a batch of fresh oranges and divided it into
100 cm3 samples. Three samples were kept at 4 °C and three samples were kept at 30 °C. The vitamin C
content of each sample was measured immediately after extraction and then each day for the next five
days.
A copy of the entries in their laboratory notebook is shown below.

(a) (i) Prepare a table and organise the data in a suitable way so that the effect or temperature on the
vitamin C content of orange juice can be displayed. (4 marks)
(ii) Use the information in your table to present the information in a suitable graphical form. (4 marks)
(b) What conclusions can be drawn from the results of this investigation? (2 marks)

(Total 10 marks)
Factors to be controlled
- Volume and concentration of DCPIP
- Concentration of Vitamin C solution
- Extent of shaking of the test tube with DCPIP must be standardised

During extraction of Vitamin C juice from fruits or vegetables,

- The mass of fruit or vegetable tissue and the volume of distilled water used for making a pulp
must be standardised.
- The conditions of storage of the fruits and vegetables must be standardised.
- The duration of storage (age) must also be standardised.

Compiled by Stafford Valentine Redden 7

Note: A less reliable method would be to count the number of drops of the vitamin C solution and juice
needed to decolourise the DCPIP.

This method is less reliable because the size of drops could be highly variable in volume.
CORE PRACTICAL THREE
Describe how membrane structure can be investigated practically, eg by the effect of alcohol
concentration or temperature on membrane permeability.
The colour of beetroot is due to the presence of a red pigment, anthocyanin, in the cell sap. You are
going to investigate the effect of temperature on the selectively permeable membranes of beetroot.

Safety
TAKE CARE WITH CORK BORER AND MOUNTED NEEDLE – MIND YOUR FINGERS
WEAR GOGGLES ONCE THE BUNSEN BURNER IS LIT
1. Use a cork borer to cut cylinders of fresh beetroot tissue. Place on a tile and cut into 3 mm wide discs.
2. Place all the discs in a small beaker and wash under a running tap for at least 5 minutes.
3. Meanwhile, label the test tubes - 30C, 40C, 50C, 60C, 70C, 80C and 100C and use a graduated
pipette to add 6 cm3 cold water to each.
4. Prepare a water bath using a large beaker, tripod and gauze and Bunsen burner.
5. Heat gently to 30C and remove the Bunsen burner.
6. Gently pick 6 beetroot discs with a forcep, one by one.
7. Place the discs in the water bath for exactly 1 minute. Then drop them into the test tube labelled 30C.
8. Leave the discs in the test tubes for at least 20 minutes.

9. Repeat the procedure for the other tubes.

10. Shake the tubes, hold to the light and compare the colour of the liquids in each.
11. If possible, use a colorimeter to compare the colours of each liquid.

SAQ8 Amir decided to investigate the permeability of beetroot cell membranes. Beetroots are root
vegetables. They appear red because their cells contain a water soluble red pigment in their vacuoles,
which cannot pass through membranes. He carried out an experiment to investigate the effect of
temperature on the permeability of beetroot membrane. High temperature disrupts the structure of the
membranes. Several beetroot discs were cut of equal dimensions. Each disc was rinsed in distilled water
3
and dried using absorbent tissue. One beetroot discs was then placed in a tube containing 25 cm of
0 0
distilled water and left for 30 minutes at 20 C. The procedure was repeated for temperatures of 30 C,
0 0 0 0
40 C, 50 C, 60 C and 70 C. After 30 minutes, each beetroot disc was removed from the water. The water
had become red and the discs were slightly pink. Each coloured solution was stirred and a sample
removed and placed in a colorimeter. The intensity of red coloration (absorbance) was determined by the
colorimeter. The experiment was repeated three times.
The results of Amir’s investigation are shown in the table below.

Temperature / oC Intensity of red colouration (absorbance) / Arbitrary units

Trial one Trial two Trial 3 Mean
20 0.2 0 0.1
30 0.4 0.3 0.3
40 0.5 0.6 0.6
50 0.9 1.0 1.1
60 1.6 1.6 1.7
70 1.8 1.6 1.8

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a) i) Suggest why it was necessary to rinse the beetroot discs before they were added to the distilled
water. (1)
ii) Suggest how the choice of temperatures could reduce the sensitivity of the results. (2)
iii) Suggest a reason for the appearance of red colour in the distilled water at 20 0C. (1)
iv) State how Amir ensured the reliability of his data. (2)
v) Give one other way in which the reliability of the data can be increased. (1)
b) Calculate the mean Absorbance in arbitrary units after 30 minutes for each temperature. Write your
answers in the spaces provided in the table. Show your working in the space below. (3)
c) Use these data to plot a fully-labelled graph to show the effect of temperature on the permeability of the
membrane. (4)
d) In order to get some idea of the validity of his data, Amir searched the Internet for similar studies. He
could found similar studies that had used his method. He compared the results from one such study,
shown in the following table, with his own.
Temperature (°C) Mean Absorbance (arbitrary units)
20 0.0
25 0.2
30 0.3
35 0.4
40 0.7
45 1.2
50 1.8
55 1.8
60 1.8
i) State one similarity and one difference in the conclusions Amir could make about the effect of
temperature on permeability of the membrane, based on these two sets of data. (2)
ii) Suggest one explanation for the similarity and one explanation for the difference you have given above.
(4)
CHSE First Semester 2008

SAQ9 The diagram below shows the fluid mosaic model of the cell membrane. (2)

(a) Name the structures labeled A. B, C and D.

(b) Describe two ways in which hydrophilic molecules, such as glucose, are able to pass through intact
membranes. (3)
(c) The graph below shows the results or an experiment to investigate the effect of temperature n the
permeability of beetroot cell membranes. The intensity of the color in the water surrounding the beetroot
was measured at the temperatures indicated oil the graph.

Compiled by Stafford Valentine Redden 9

(i) Outline a procedure that you could have used to produce these results. (4)
(ii) Explain the effect or the increase in temperature on the permeability of the membranes of beetroot
cells. (3) Total 12 marks SNAB Jan 2005

SAQ10 Beetroots are root vegetables. They appear red because their cells contain a water soluble red
pigment in their vacuoles, which cannot pass through membranes. An experiment was carried out to
investigate the effect of alcohol on the permeability of beetroot membrane. Alcohol disrupt the structure of
the membranes. Several beetroot discs were cut of equal dimensions. Each disc was rinsed in distilled
3
water and dried using absorbent tissue. Five beetroot discs were then placed in a tube containing 25 cm
0
of 0.2% ethanol and left for 30 minutes at 20 C. The procedure was repeated for different concentrations
of ethanol and one set of discs was left in distilled water. After 30 minutes, each set of beetroot discs was
removed from the solutions and from the water. The ethanol in each tube had become red and the discs
were slightly pink. There was no change in the colour of the discs in the water and the water remained
colourless. The ethanol in each tube was stirred and a sample removed and placed in a colorimeter. The
intensity of red coloration (absorbance) was determined by the colorimeter. The results of the investigation
are shown in the graph below.

a) Explain why the cell membrane is described as having a fluid mosaic structure. (2)
b) Suggest why it was necessary to rinse the beetroot discs before they were added to the bile salt
solution. (1)
Compiled by Stafford Valentine Redden 10
c) Describe the effect of increasing ethanol concentration on the intensity of the red colour (absorbance) of
the solution. (3)
d) Suggest an explanation for these results. (4)
e) The experiment was repeated using a second beetroot. Suggest why the readings obtained might be
slightly different from those for the first beetroot. (1) SVR CHSE 2009

Working of the colorimeter – Cuvette contains pigment solution

Factors to be controlled
- The storage conditions and age of beetroot must be taken into account
- The diameter and thickness of the discs must be standardised
- The volume of water or ethanol in the test tube must be controlled
- The number of discs in the test tube must be the same for all trials
- The temperature of the water bath must be controlled
- The duration of temperature or ethanol treatment must also be standardised
- The volume of red pigment in the cuvette must be standardised
- A blue filter must be used in the colorimeter during measurement of absorbance

CORE PRACTICAL FOUR

Describe how enzyme concentrations can affect the rates of reactions and how this can be
investigated practically by measuring the initial rate of reaction.

Investigating an enzyme controlled reaction: catalase and hydrogen peroxide concentration

Procedure
SAFETY: Wear eye protection and protect clothing from hydrogen peroxide. Rinse splashes of peroxide
and pureed potato off the skin as quickly as possible as it is an oxidising agent.

Investigation
a Use the large syringe to measure 20 cm3 pureed potato (pulp) into the conical flask.
b Put the bung securely in the flask – twist and push carefully.
c Half-fill the trough, bowl or sink with water.
d Fill the 50 cm3 measuring cylinder with water. Invert it over the trough of water, with the open end
under the surface of the water in the bowl and with the end of the rubber tubing in the measuring
cylinder. Clamp in place.
Compiled by Stafford Valentine Redden 11
e Measure 2 cm3 of hydrogen peroxide into the 2 cm3 syringe. Put the syringe in place in the bung of the
flask but do not push the plunger straight away.
f Check the rubber tube is safely in the measuring cylinder. Push the plunger on the syringe and
immediately start the stop clock.
g After 30 seconds, note the volume of oxygen in the measuring cylinder in a suitable table of results.
h Empty and rinse the conical flask and measure another 20 cm3 pureed potato into it. Reassemble the
apparatus, refill the measuring cylinder, and repeat from d to g with another concentration of hydrogen
peroxide. Use a 100 cm3 measuring cylinder for concentrations of hydrogen peroxide over 20 vol.
i Calculate the rate of oxygen production in cm3/ s.
j Plot a graph of rate of oxygen production against concentration of hydrogen peroxide.

Enzyme Kinetics
This means measuring the rate of enzyme reactions.
1. Firstly you need a signal to measure that shows the progress of the reaction. The signal should
change with either substrate (S) or product (P) concentration, and it should preferably be something that
can be measured continuously. Typical signals include colour changes, pH changes, mass changes, gas
production, volume changes or turbidity changes. If the reaction has none of these properties, it can
sometimes be linked to a second reaction, which does generate one of these changes.

2. If you mix your substrate with enzyme and measure your signal, you will obtain a time-course. If
the signal is proportional to substrate concentration it will start high and decrease, while if the signal is
proportional to product it will start low and increase. In both cases the time-course will be curved (actually
an exponential curve).

3. How do you obtain a rate from this time-course? One thing that is not a good idea is to measure the
time taken for the reaction, for as the time-course shows it is very difficult to say when the reaction ends: it
just gradually approaches the end-point. A better method is to measure the initial rate - that is the initial
slope of the time-course. This also means you don't need to record the whole time-course, but simply take
one measurement a short time after mixing.

4. Repeat this initial rate measurement under different conditions (such as different substrate
concentrations) and then plot a graph of rate vs. the factor. Each point on this second graph is taken from
a separate initial rate measurement (or better still is an average of several initial rate measurements
under the same conditions). Draw a smooth curve through the points.

Be careful not to confuse the two kinds of graph (the time-course and rate graphs) when
interpreting your data.

An example of time course measurement is shown in the SAQ below.

SAQ11 The total amount of product formed in an enzyme - controlled reaction was investigated at two
different temperatures, 55 °C and 65 °C. The results are shown in the graph.

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a) (i) Explain how you would calculate the rate of the reaction at 55°C over the first 2 hours of the
investigation. (1)
(ii) Explain why the initial rate of this reaction was faster at 65 °C than it was at 55 °C. (3)
b) Use your knowledge of enzymes to explain the difference in the two curves between 4 and 6 hours. (1)
c) In this investigation, the enzyme and its substrate were mixed in a buffer solution. What was the
purpose of the buffer solution? (1) (Total 6 marks)

An example of initial rate measurement reaction is shown in the SAQ below.

SAQ 12 The graph below shows the results of an investigation into the effect of substrate concentration
on the initial rate of enzyme controlled reaction.
0.20
B C

Initial 0.15
rate of
reaction
/ mg of
products s –1
0.10

0.05

0
0 10 20 30 40 50 60
Substrate concentration / mg cm –3
a) Suggest two conditions apart from temperature that should be kept constant in this investigation. (2)
b) Explain why changes in the substrate concentration cause an increase in the rate of reaction between
points A and B on the graph. (2)
c) Suggest why the curve levels off between points B and C. (2)
Compiled by Stafford Valentine Redden 13
d) On the graph, sketch a curve to show how the results for the investigation would change if it were
repeated at a lower temperature. Explain any difference between the two curves. (3)
(Total 9 marks) January 2002 Unit 1, Edexcel
SAQ13 A student carried out an experiment to test the hypothesis that an increased concentration of the
substrate increases the initial rate of an enzyme-controlled reaction.
For this investigation, hydrogen peroxide was used as the substrate, and liver tissue was used as the
3
source of the enzyme catalase. The enzyme was extracted by breaking up 3.0g or liver in 250cm of water
3 -3
in a blender for 45 seconds. At the start. 20cm of 31.7 mmol dm hydrogen peroxide was placed in a
beaker. One 6mm diameter disc of filter paper. previously soaked in Iiver extract. was then put at the
bottom of the beaker and a stop watch as started. Oxygen bubbles, produced as a result of the reaction,
caused the disc to rise to the surface of the liquid in the beaker. The time in seconds for the disc to reach
the surface was recorded. This procedure vas repeated for two more discs and the mean time was
recorded. The experiment was then continued using increasing concentrations of hydrogen peroxide.
A copy of the student's laboratory notes showing the mean times for the discs to rise to the surface at
each concentration is given below.

(a) The initial rate or reaction can be calculated using the formula below.
Initial rate = 1 / Mean time
Prepare a table and present the data to show the initial rate of reaction at each concentration of hydrogen
peroxide. (4)
(b) Use the data in your table to present the information in a suitable graphical form. (4)
(c) Describe the trends and patterns shown by these data. (2)
(d) What conclusions regarding the hypothesis can be drawn from this investigation? (2)
(e) Describe two limitations of this technique that could give rise to unreliable results. (2)
(f) Compare the reliability of this method with the method describes in the core practical described
above and give reasons for your answer.
(g) Describe how a solution containing 160 mmol of hydrogen peroxide per dm3 would be diluted
to prepare a solution containing 80 mmol of hydrogen peroxide per dm3. (2 ) SVR

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 CORE PRACTICAL FIVE

Describe the stages of mitosis and how to prepare and stain a root tip squash in order to observe
them practically.

Method
PROCEDURE:
1. Place some 1 molar Hydrochloric acid in the watch glass. Be careful not to get the acid on your skin or
clothing.
2. In to this acid place the terminal 3 or 4 mm of the 1 cm long onion root.
3. In a short time (a few minutes) the root tip will feel soft when touched with a dissecting needle.
4. Now, using forceps or a needle, pick up the softened root tip and transfer it in to a drop of acetocarmine
stain on a clean slide.
5. Using a razor blade or a sharp scalpel, shop the root tip in to tiny pieces. Note: Iron in the scalpel or
dissecting needle reacts with the acetocarmine stain to give a better staining reaction.
6. Once this procedure is complete, apply a clean cover slip to the slide and heat it gently over an alcohol
lamp or slide warmer. Do not boil. Then invert the slide on a blotting paper and push down ward firmly,
applying pressure with your thumb over the cover glass. This should flatten the cells and disperse them so
they can be observed under the microscope.

SAQ14 (a) The table below describes some of the key events that occur during mitosis Complete the
table by writing the name of the stage of mitosis next to its description.
Key events Stage
Chromatids separate and move to opposite poles of the dividing cell.
Chromosomes shorten and thicken. The nuclear envelope breaks down and the spindle forms.
The spindle fibres break down, the nuclear membrane re-forms and the chromosomes
elongate.
Chromosomes line up on the equator of the cell, attached to spindle fibres by their
centromeres.
(4)
(b) The graph below illustrates the change in DNA content during the cell cycle

Mass of DNA /
arbitrary units
6
C D
5

3 A B

2
G1
1

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time / hours

(i) Calculate the percentage of the cell cycle time spent in G1. (3)
(ii) At which point, A, B, C or D, does chromosome replication (the S phase) begin? Explain your answer.
(Total 9 marks)
SAQ 15 The diagram below shows cells from a root tip, prepared by the root tip squash method.

Compiled by Stafford Valentine Redden 15

(a) Describe how you would prepare a root tip squash so that mitosis can be studied. (4)
(b) State which of the cells labelled A–E is in:
(i) metaphase ...................
(ii) anaphase ..................... (2)
(c) State two events that take place during interphase. (2) (Total 8 marks)

SAQ 16 The photograph shows a cell in mitosis as viewed using a transmission electron microscope.

(a) The actual diameter of the cell between points X and Y is 50 µm. Calculate the magnification of this
photograph. Show your working. (3)
(b) In the space below make an accurate drawing of the cell. Label the chromosomes, spindle fibres and
centrioles. (5)
(c) State the stage of mitosis that this cell is in. (1)
(d) State one function of each of the following.
Spindle fibres
Centrioles (2) (Total 11 marks)

SAQ 17 The diagrams below show four stages of mitosis.

Compiled by Stafford Valentine Redden 16

 A B

C D
(a) (i) Write the letters of the stages in the sequence in which they occur during mitosis. (1)
(ii) Name stage D. (1)
(b) The graph below shows how the quantity of DNA varies with time in a cell cycle.

Quantity of DNA
/ arbitrary units
3

0
0 5 10 15 20 25
Time/hours
(i) Explain the changes in the quantity of DNA that take place:
between 10 to 15 hours
at 20 hours (2)

(ii) What is happening - in the cell between 15 and 20 hours? (1)

(iii) What is the minimum length of time that interphase would occupy in this cell cycle? (1)
(Total 6 marks)

SAQ 18 The flow diagram below shows a method for preparing and staining cells in order to study stages
of mitosis.

Compiled by Stafford Valentine Redden 17

 Treat plant material with hydrochloric acid

Place in stain and warm

Break open plant material

Mount on slide

Squash gently

(a) Name a suitable part of a plant to use, giving a reason for your choice. (2)
(b) (i) Explain why staining is necessary in this preparation. (1)
(ii) Name a suitable stain for this technique. (1)
(c) Explain why it is necessary to squash the preparation. (1)
(Total 5 marks)

SAQ19 An investigation was performed to determine the length of time that a cell in an onion root tip
spends in each stage of mitosis.

A growing root from an onion was selected and a root tip squash was made. This was examined under a
light microscope and the percentage of cells in each stage of mitosis was determined.

The results are shown in the table below.

Percentage of cells in this

Stage of mitosis
stage
Prophase 2.43
Metaphase 1.40
Anaphase 0.70
Telophase 2.78

(a) Describe how you would prepare a root tip squash so that mitosis could be studied. (4)

(b) The percentage of cells in a stage of mitosis is proportional to the duration of that stage. Use this
information to compare the duration of each stage of mitosis in these root tip cells by drawing a suitable
graph. (3)

(c) The duration of each stage of mitosis can be calculated using the equation below.

Percentageof cells in thatstage  cell cycle time

Duration of a stage =
100

Compiled by Stafford Valentine Redden 18

The cell cycle time for these cells is 1200 minutes.
Determine the duration of each stage of mitosis and present the data in a suitable table. (2)
(Total 9 marks)

SAQ 20 The diagrams below show some cells in different stages of mitosis.

A
B

(a) Name the stages of mitosis shown by the cells labelled A, B and C. (3)
(b) Describe the events that occur in the stage of mitosis shown by cell D. (2)
(c) Explain the significance of the stage shown by cell D. (1) (Total 6 marks)

SAQ21 The diagram below shows the stages in the cell cycle of a plant root cell.

Cytokinesis

Telophase Interphase
Anaphase
Metaphase

Prophase

(a) The cell had 2 arbitrary units of DNA at the start of interphase. State the number of arbitrary units of
DNA in this cell in each of the following stages.
(i) at the end of prophase (1)
(ii) during anaphase (1)
(b) Describe how you would prepare a root tip squash to observe the stages of mitosis. (5)

Compiled by Stafford Valentine Redden 19

(c) Two onion bulbs were grown to obtain some roots. One onion bulb was grown in water, the other in a
solution of a drug called vincristine, as shown below.

Onion bulb

Roots

Water Solution of
vincristine

Vincristine is a drug used in the treatment of cancer. It prevents spindle formation during mitosis. The
result of a root tip squash on the roots grown in a solution of vincristine showed an increase in the
percentage of cells found in one of the phases compared with roots grown in water.
(i) Suggest in which phase of mitosis this increase occurs. (1)
(ii) Give an explanation for your answer. (2)
(iii) State the precautions that must be taken for the results of the comparison to be reliable. (4)
iv) Use the information in the pie chart to draw a bar graph, showing the relative length of each stage in
the cell cycle. (4) SVR (Total 18 marks)

CORE PRACTICAL SIX

Describe how totipotency can be demonstrated practically using plant tissue culture techniques.
Tissue culture techniques for the vegetative propagation of plants (micropropagation) have a number of
advantages over other methods.
 Growth is very rapid.
 Growth is independent of seasons.
 The technique can be applied to a number of species that are otherwise difficult to propagate.
 Apical meristems in infected plants often remain virus-free. Their use for tissue culture has, therefore,
permitted the elimination of viruses from infected stocks of a range of species.
 Certain types of callus culture give rise to clones that have inheritable characteristics different from
those of the parent plant. Improved varieties arising in this way can be propagated and used
commercially.
 In the future, it is likely that new varieties and hybrids produced using other modern techniques will be
commercially propagated by tissue culture methods.
 A recent development has been the commercial use of callus cultures of the shikon plant to produce
large quantities of shikonin, a medically valuable product found, in a lower concentration, in the shikon
root. Plants are valuable sources of many such products, and the application of tissue culture techniques
in this way may, therefore, become more economically important.
Procedure
Collect all the apparatus that you will need and prepare any solutions that are required. Set up and
sterilize your work-bench as suggested in the notes on aseptic handling techniques.
1. Select a clean floret from a fresh cauliflower head. Place it on a tile and. holding it with forceps.
Carefully trim off mini-fIorets' from the curd to produce 20 cuboids’ of curd tissue, approximately 3 mm x
5mm x 5 mm. These will be your explants.
Compiled by Stafford Valentine Redden 20
The next stage of the procedure is to steriiize the surface of the explants with a chlorate (I) solution
(Bleach).
Caution: You will be ushig a fairly strong bleach solution so take extreme care. From now on you
must use aseptic handling techniques (p. 17), so return your forceps to the ethanol beaker.
2. Quickly transfer your explants to a clean (preferably sterile) screw-top jar and add chlorate (l) solution to
leave a small head space. Reseal the jar and shake the contents for five seconds.
3. Shake the jar for five seconds every minute for exactly 10 minutes.
4. After exactly 10 minutes. pour off the hypochlorite solution into the 'waste’ beaker, using the Jar lid to
trap the sterilized explants.
3
5. wash the explants four times as follows. Add approximately 100cm sterile water to the jar. Reseal it,
shake for five seconds and pour off the liquid into the 'waste' beaker as in Step 4. The explants may be left
in the last wash until they are required.
6. Using sterile forceps (cooled in the wash water) transfer six explants to each of the three petri dishes
containing growth medium. The explants should be widely spaced and pressed gently onto the agar.
Flame sterilize and cool your forceps when each dish is complete.
7. Seal each dish with Parafilm or insulating tape to reduce dehydration.
8. Label each dish clearly on its base and incubate them in the light at 200C to 280C.
9. Examine each culture weekly. record and sketch any changes you observe, If some of the explants
show signs of contamination, the remainder should be aseptically transferred to fresh medium (Steps 6—
8).

Aseptic techniques – the systematic precautions taken to keep cultures pure and free from
contamination.
 All apparatus (Petri dishes, pipettes, flasks, etc) must be sterilised by autoclaving at 121 0C for 15 minutes at
103 kPa. Or by irradiating with ultraviolet rays (Wavelength - 254nm).
 Sterile the culture medium by autoclaving.
 Wipe the bench / work surfaces with 70% ethanol before and after work.
 Flame the necks of bottles to prevent air-borne contamination.
 Flame wire loops and forceps before and after use or sterilise by dipping in ethanol.
 Lids of containers should not left on benches.
 Petri dishes opened slightly during operation to avoid contamination.
 Cultures after study and contaminated equipment must be autoclaved / sterilised before disposal or reuse.

The composition of the medium used for growth is shown in the table below.
Growth substances used in tissue culture
experiments
Phytochromes – Photosensitive pigments..
Caution: Some phytohormones are toxic, so
care must be taken when preparing stock
solutions, e.g. protective gloves should be
worn. Students should not, of course, come
into direct physical contact with media.
Auxins (Stimulate root development)
e.g Indol 3-yl acetic acid (IAA)
1-Naphthyl acetic acid (NAA)
2,4-dichlorophenoxy acetic acid (2,4-D)

Cytokinins (Stimulate cell division)

6-furfurylaminopurine (Kinetin)
6-benzylaminopurine (BAP)

Compiled by Stafford Valentine Redden 21

 Outline of plant tissue culture

Compiled by Stafford Valentine Redden 22

SAQ 22 Plant tissue culture is a method used to propagate plants. The flow diagram shows one method of
plant tissue culture.

(a) Name the type of reproduction involved in plant tissue culture. (1 mark)
(b) Give two advantages of producing plants using this method rather than from seeds. (2 marks)
(c) Why is a viral infection more likely to destroy a complete batch of plants grown by plant tissue culture
than a batch of plants grown from seeds? (1 mark)
(d) Callus tissue develops into either shoots or roots depending on the relative concentration of the plant
growth regulators used. Use your knowledge of genes to suggest how these plant growth regulators
determine the type of plant tissue formed. (1 mark)
Compiled by Stafford Valentine Redden 23
(e) The data below shows the growth of five callus’ grown at different concentrations of sucrose in the
culture medium.

Sucrose
-3 -3 -3 -3 -3
concentration 0.1 mmol dm 0.2 mmol dm 0.3 mmol dm 0.4 mmol dm 0.5 mmol dm
Initial mass
of callus 2.1 g 2.6g 2.3g 2.1g 2.7g
Final mass
of callus 2.6g 3.8g 4.6g 4.2g 5.4g
(After one week)

i) Prepare a table and organise the data in a suitable way, so that the growth of the callus at different
sucrose concentrations can be compared. (4)
ii) Present your data in a suitable graphical form. (4)
iii) Suggest why the reliability of the data may be low. (3)
iv) List the factors that must be controlled to get reliable results. (4)
SVR

SAQ 23 ‘Take-all’ is a disease of wheat caused by a fungus. It can cause serious damage to the crop.
There is no gene for resistance to this fungus in wheat. There is, however, a gene for resistance to this
fungus present in oats.
The diagram shows how this gene might be transferred to wheat.

(a) (i) The wheat plant with the resistance gene contains recombinant DNA. What is recombinant DNA?(1)
(ii) The plasmids act as vectors for the resistance gene. What is a vector? (1)
(iii) Suggest how cells with the resistance gene might be selected. (2)
b)i) Suggest which tissue would be most suitable for the explants. (1)
ii) Explain why explants containing large quantities of xylem do not usually grow into a callus. (2)
c) State what is meant by the term totipotency. (2)
d) Distinguish between differentiation and dedifferentiation. (2)
e) Suggest why adult animal cells usually do not develop into complete organisms, but plant tissues do.(2)
SVR

Compiled by Stafford Valentine Redden 24

 CORE PRACTICAL SEVEN
Describe how to determine the tensile strength of plant fibres practically.
Extracting fibres from plants
The objectives of this procedure are:
 to extract fibres with possible commercial use from plants
 to develop investigative and experimental skills: devise a hypothesis, plan an experiment to produce
appropriate results to test that hypothesis, use apparatus and a procedure that is suitable to produce
valid results.

Introduction

Fibres have been extracted from plant stems for centuries and used in the commercial manufacture of a
wide range of textiles and paper. The term ‘fibres’ is used to describe a range of ‘fibre-like’ structures, not
just the sclerenchyma. The use of different fibres depends on their properties.

Strength can be defined as the maximum stress a material can withstand without failing (breaking).
Tensile strength is the maximum stress caused by a pulling force that a material can stand without failing.
Concrete has a tensile strength of 2 x 106 N m-2. Compression strength is the maximum strength caused
by a pushing force that a material can stand without crushing.

Plant stems must not only be strong. Often they must be able to bend in the wind and return to their
original shape without any permanent distortion. They must not be too stiff.

Fibre Useful part of the plant Applications

Flax Stem of flax plant Linen for clothing
Cotton Hairs on the seeds of a plant belonging to the Cotton for clothing
mallow family
Hemp Fibres from stem/ leaves of the hemp plant Used for ropes and carpet-backing
Coir Fibre from the husks of the fruit of the coconut Floor coverings, ropes
Jute Fibre from the stem of the jute plant Hessian – sacking and carpets
Manila Hard fibres from the leaves of a type of Marine cables and other ropes, nets
banana and matting
Pulp Softwood trunks Paper, cardboard
Fibres can be removed from the plant stems by retting. In field retting, the plant stems are cut or pulled up
and left to rot in the field where microbial action breaks down the stalks. In water retting, stems are
immersed in water. During soaking, bacteria and fungi break down the soft tissues of the stems leaving
the cellulose intact. This produces more uniform, higher quality fibres, but is more expensive and
produces nitrogen-rich waste water that must be treated before discharge. It is relatively easy to remove
the cellulose-rich fibres, as described below.
Procedure
SAFETY: Wear eye protection and gloves when handling the plant material.
Wash your hands after handling the soaked material.
When testing fibres to breaking point, make sure the loads on the material can fall without causing injury.

Preparation of plant fibres

Remove the stems from the water. Wash the stems to remove the softened tissue and then dry the
remaining fibres on a paper towel. The outside cuticle and epidermal layer will rub away and the central
pith will be left when you peel away the fibres. These fibres are made up of vascular tissue; they contain
both the xylem vessels and sclerenchyma fibres.

Compiled by Stafford Valentine Redden 25

SAQ24 Sisal is a material used to make rope. It is made from the sclerenchyma fibres found in the leaves
of the plant, Agave sisalana.
After extraction of the fibres, the waste leaf material can be used in the production of organic fertiliser.
The four countries that produce most of the world’s sisal are Brazil, Kenya, Tanzania and Madagascar.
The table below shows the annual harvest of freshly-cut Agave sisalana leaves together with the total
annual production of sisal.

(a) (i) Complete the table to show the total annual production of sisal. (1)
(ii) Calculate the total percentage of sisal produced from freshly-cut leaves. Show your working. (2)
(b) Nylon is a synthetic (man-made) fibre which can be used to make ropes. Nylon ropes are lighter and
stronger than those made using sisal. Suggest two advantages of using sisal rather than nylon to make
ropes. (2)
(c) (i) Explain what is meant by the term tensile strength of a fibre. (1)
(ii) Suggest how you could carry out a practical investigation to compare the tensile strength of sisal and
nylon fibres. (4)
1. idea of suspending {fibres / bundles of fibres} with weights on / pulling them with a
forcemeter/eq;
2. { fibres / bundles of fibres} of same diameter used / different diameters accounted for;
3. { fibres / bundles of fibres} of same initial length used;
4. detail of how {weights added / forcemeter pulled};
5. description of measurable endpoint eg {breaking point / stretched to standard length};
6. repeated readings taken at each {weight / forcemeter reading} (using different fibre NOT just
checking the reading with same fibre);
7. reference to a safety procedure eg {goggles in case fibre snaps / precaution against falling
weights};
8. ref to control of {temperature / humidity / other relevant factor};

(d) Describe two ways in which the structure of xylem vessels is similar to that of sclerenchyma fibres. (2)

Compiled by Stafford Valentine Redden 26

 CORE PRACTICAL EIGHT
Describe how to investigate plant mineral deficiencies practically.
The aim of this practical is to investigate the effects of mineral deficiency on the growth of plants.
Seedlings are grown in solutions containing a range of mineral salts, including those which lack
phosphate, nitrate, calcium, potassium, magnesium, iron and sulphate, plus the complete medium.

Suitable seedlings which can be used for this experiment include: maize (Zea mays), castor beans
(Ricinus communis), tomato (Lycopersicon esculentum) and cabbage (Brassica oleracea). Seeds are
germinated in moist vermiculite (a nutrient free medium that retains moisture and anchors roots at the
same time), then transferred to the nutrient solution, as shown in the Figure. At weekly intervals, the plants
are measured and any deficiency symptoms noted.
Method
1. Make up the culture media and set up eight tubes containing the following:
 Complete (normal) medium
 Medium lacking phosphate
 Medium lacking nitrate
 Medium lacking calcium
 Medium lacking potassium
 Medium lacking magnesium
 Medium lacking iron
 Medium lacking sulphate
2. Wrap each tube in aluminium foil.
3. Select eight seedlings and set up the cultures as shown in Figure.
4. Leave the experiment at room temperature and each week record the following:
 Shoot and root length
 Leaf number and size
 Internode number and length
 Deficiency symptoms, such as changes in shape or yellowing of leaves.

SAQ 25 The apparatus shown below can be used to study mineral nutrition in flowering plants.

(a) Suggest the function of each of the following.

(i) The stream of air (2)
(ii) The black paper cover (2)

(b) The nutrient solution contains various mineral ions including magnesium and phosphate. Give
one reason why each of these ions is essential to the plant.
Magnesium
Phosphate (2) (Total 6 marks)

Compiled by Stafford Valentine Redden 27

SAQ26 An investigation was carried out into the absorption of mineral ions by beech tree seedlings.
The absorption of phosphate ions by beech roots was measured in moist air, and in an atmosphere
of moist nitrogen.

The results are shown in the graph below.

10

Absorption of 6
Roots in air
phosphate ions
Roots in nitrogen
/ arbitrary units 5

0
0 5 10 15 20 25 30
Time / hours

(a) Calculate the rate of absorption of phosphate ions by beech roots in air between 10 hours and 20
hours. Show your working. (3)
(b) (i) Compare the rates of absorption of phosphate ions by roots in air and roots in nitrogen. (2)
(ii) Suggest an explanation of the difference in rates of absorption. (2)
(c)(i) Suggest two reasons why the atmosphere in which the roots are kept has to be moist. (2)
(ii) Suggest two factors that should be kept constant in this experiment. (2) (Total 11 marks)

Compiled by Stafford Valentine Redden 28

SAQ 27 An experiment was carried out to investigate the uptake of different mineral ions by barley
plants. A large number of barley seedlings was grown in a nutrient solution containing a range of
mineral ions including potassium (K+), calcium (Ca2+), magnesium (Mg2+) and nitrate (NO3–).

The experiment was set up as shown in the diagram below.

The concentration of these ions in the solution were measured at the beginning and at the end of the
experiment.

The results are shown in the table below.

Concentration of ions in nutrient solution / arbitrary units

Mineral ion
At start of experiment At end of experiment
Nitrate 7.0 1.8
Potassium 3.0 0.0
Magnesium 2.0 2.1
Calcium 5.0 5.6
(a) Calculate the percentage difference between the concentration of nitrate ions at the beginning
and at the end of the experiment. Show your working. (3)
(b) What do the results suggest about the mechanism of absorption of potassium ions?
Explain your answer. (3)
(c) Suggest an explanation for the changes in concentrations of magnesium and calcium ions during
the experiment. (2)
(d) State two precautions which should have been taken to ensure that results for all the barley
seedlings were comparable. (2)
(e) Describe the pathway taken by mineral ions as they pass from the nutrient solution to the xylem
in the roots of the seedlings. (3)
(Total 13 marks)

Compiled by Stafford Valentine Redden 29

SAQ 28 Duckweeds are small, green plants that float on the surface of freshwater ponds.
Experiments were carried out on the growth of two species of duckweed, Lemna gibba and Lemna
polyrrhiza. L. gibba plants contain non-photosynthetic air sacs which enable them to float on the
surface of the water.
In these experiments the two species were grown in complete mineral nutrient solutions as follows.
Experiment A Lemna gibba and Lemna polyrrhiza together
Experiment B Lemna polyrrhiza only
Experiment C Lemna gibba only
The dry mass of the plants was measured each week for 8 weeks.
The results for experiment A are shown in the table below.
Dry mass / mg
Week
Lemna polyrrhiza Lemna gibba

0 25 22
1 69 84
2 87 169
3 106 197
4 87 289
5 100 344
6 87 344
7 81 347
8 37 345

(a) Plot the data in suitable graphical form on graph paper. (5)
(b) Describe the growth of L. polyrrhiza and L. gibba in experiment A. (4)

(c) The results for experiments B and C are shown in the table below.
Week Experiment B Experiment C
Lemna polyrrhiza Lemna polyrrhiza
0 63 50
1 100 119
2 188 200
3 306 259
4 381 291
5 448 356
6 562 350
7 594 369
8 641 400
(i) Compare the growth of L. polyrrhiza in experiments A and B (2)
(ii) Suggest a reason for the difference in growth in these two experiments. (1)
(d) (i) How does the growth of L. polyrrhiza in experiment B differ from L. gibba in experiment C?
(ii) Suggest an explanation for this difference. (2) (Total 15 marks)

Compiled by Stafford Valentine Redden 30

 CORE PRACTICAL NINE
Describe how to investigate the antimicrobial properties of plants.

Preparation of media and plate pouring

Introduction
There are many different types of media which are used for the culture of micro-organisms. These may be
obtained in ready-formulated preparations, or can be made using separate ingredients. In this practical,
we look at the method for preparing and sterilizing nutrient agar medium.
Nutrient agar medium can be used for the culture of bacteria. If ready-formulated media are used, then it
is made up using distilled water.
Materials
 Ready-formulated Nutrient agar medium.
 Distilled water
 50 ml Universal bottle
 Petri dishes
 Autoclave
 Incubator
 Gas burner
 Glass rods
 Electronic weighing balance
 Measuring cylinders
 Conical flasks, sterilized cotton

Method
Nutrient agar medium
Add 28 g of formulated ingredients in 1 litre of distilled water and stir it until it dissolve.
Dispense into a suitable containers and autoclave at 121 ºC for 15 minutes.

Procedure for pouring a plate

Allow liquid agar to cool to about 50 ºC (just cool enough for comfortable handling) after removal
from autoclave. Unscrew cap of bottle with little finger as shown in Figure (a). It can be held by little
finger during the procedure to avoid placing it on the bench where it could become contaminated.

‘Flame’ mouth of bottle for a few seconds. This is done to produce an upward flow of air from the
bottle so that any organisms in the area will not fall into the bottle. (It is not done to kill micro-
organisms; the bottle would have to be heated for too long to achieve this effect). Refer Figure (b).

Pour the molten agar (about15-20 cm3) slowly into the base of the sterile Petri dish, lifting the lid
only as much as necessary. Do not spill agar on the edges of the dish (if so, start again). Replace
the lid and allow the agar to set. Refer Figure (c).

If the plate is to be used for ‘streaking’ or ‘spreading’ the surface of the agar and lid can be dried
once the agar is set. They are best dried upside down in an incubator at about 37 ºC, and
arranged as shown in Figure (d) to minimize the risk of contamination (slight in these
circumstances).
Preparation of streak plate of bacteria

Introduction

In this practical, an agar plate will be poured using sterile nutrient agar and inoculated using a culture of a
suitable bacterium, such as Bacillus subtilis. Streak plates are useful to isolate pure culture as, individual
colonies will have grown from a single cell. Single colonies can be used to subculture another agar plate
to obtain a pure isolate.

Materials
 Sterile Petri dishes
 Sterile nutrient agar
 Boiling water bath
Compiled by Stafford Valentine Redden 31
  Bacteriological loop
 Slope culture of Bacillus subtilis, or other suitable bacterium.
 Chinagraph pencil or spirit marker pen

Method
Pouring a sterile agar plate
1. Before starting, wipe the bench surface using a suitable disinfectant solution
2. Melt the agar in a boiling water bath, remove carefully using tongs allow to cool to about 45
ºC. At this temperature, the agar will be cool enough to handle safely, but will remain molten.
Agar starts to set below about 42 ºC.
3. On the base of sterile Petri dish, write your name, the date and the name of the organism with
which the plate will be inoculated. Petri dishes should be always be labeled on the base, as it is
possible for lids to be transposed.
4. Working near a Bunsen burner with a blue flame, hold the molten, but cooled, nutrient agar in
one hand and using little finger of the other hand, remove the lid of the bottle. Do not place the
lid on the bench.
5. Pass the neck of the bottle through the Bunsen flame using the hand in which you are holding
the lid of the bottle, raise the lid of the Petri dish to an angle of about 45 and carefully pour in
the agar until the dish is nearly half full. Replace the Petri dish lid, flame the neck of the bottle
again and replace the lid.
6. Leave the agar plate to set.

Method
Preparing a streak plate
1. Have ready your sterile agar plate and slope culture of the bacterium to be used.
2. Sterilize the bacteriological loop by holding it in a blue Bunsen flame until red hot.
3. Allow the loop to cool and whilst still holding the loop, remove the lid from the slope culture
using the little finger of the hand in which you are holding the loop. Do not place the lid on the
bench.
4. Pass the neck of the culture bottle through the Bunsen flame, then use the loop to remove a
small portion of the culture. Replace the lid on the culture bottle.
5. Now lift the lid of the Petri dish and use the loop to streak out the culture as shown in Figure.
Be careful not to ‘Plough up’ the surface of the agar. When you have finished, flame the loop
again before placing it on the bench.
6. Fasten the lid of the Petri dish using two pieces of adhesive tape. Invert the dish, and incubate
at 30 ºC for 24 hours.

Results and discussion

1. Record the appearance of your streak after incubation. Were you successful in obtaining single
colonies of the bacterium?
2. List the different methods of sterilization, which have been used in this practical.
3. Explain why it is important:
(a) to avoid ploughing up the surface of the agar when inoculating
(b) not to seal the dishes all around with adhesive tape
(c) to invert the dishes when they are incubated.

SAQ 29 Mutation in bacteria can be investigated by inoculating agar plates containing

concentrations of antibiotic sufficiently high to prevent bacterial growth. Only bacteria that
have mutated and become resistant to the antibiotic will grow and form colonies.

An experiment was carried out to investigate the mutagenic potential of three compounds, A, B
and C. Bacteria of a strain known to be sensitive to penicillin were exposed for two minutes to
varying concentrations of compounds A or B or C. Plates containing penicillin were then
inoculated with 107 bacteria and incubated for three days.

Compiled by Stafford Valentine Redden 32

The results are shown in the table below.

Number of colonies
Concentration of compound
/ g cm–3 Compound A Compound B Compound C

0 2 1 2
10 3 4 4
20 6 9 3
30 7 14 5
40 11 23 2
50 14 34 1
100 24 7 3
200 40 0 6

(a) Describe the results obtained with each compound and in each case suggest a reason
for the result.
Compound A
Compound B
Compound C (6)
(b) Mutation rate is defined as the chance that a mutation would occur in one generation.
Calculate the mutation rate for the bacteria in compound A at 200 µg cm -3
concentration. Show your working. (2)

(c) Suggest two reasons why bacteria are suitable organisms for studies of mutation. (2)
(d) Suggest why antibiotics used to treat human disease should not be used to treat disease
in animals reared for food. (2)
(e) Draw a graph to compare the effects of the concentration of the compound A,B and C on
the growth of the colonies. (4) (Total 16 marks)

SAQ 30 An experiment was carried out to test the sensitivity of the bacterium Bacillus subtilis to
different antibiotics. The surface of a nutrient agar plate was flooded with a broth culture of the
bacterium. The excess broth was discarded. Five filter paper discs, (A, B, C, D and E), each
containing a different antibiotic, were placed on the nutrient agar surface.

The plate was allowed to stand for 30 minutes at room temperature before being incubated at
30 °C for 24 hours. The appearance of the plate after the incubation is shown in the diagram.

Bacteria growing
on agar
A

E
Clear B
zones

Antibiotics discs
D C

(a) State two ways in which antibiotics may inhibit the growth of bacteria such as
Bacillus subtilis. (2)
(b) Suggest why the surface of the nutrient agar was flooded with the broth culture rather
than being inoculated by a streaking technique. (1)
Compiled by Stafford Valentine Redden 33
 (c) Suggest why the plate was allowed to stand for 30 minutes at room temperature before
being incubated. (1)
(d) Suggest what the results indicate about the sensitivity of B. subtilis to the antibiotics. (1)
(Total 5 marks)

SAQ 31. In the culturing of microorganisms in the laboratory it is necessary to use aseptic
techniques to prevent contamination.

The diagram below shows how a simple autoclave is used to sterilise laboratory equipment
and culture media.
Valve Valve
open Mass closed
added

Steam

Aluminium
foil

Cotton
wool plug
Water
Left to cool
Heat Heat

(a) The pressure of steam builds up to 103 kPa. This raises the boiling point of water to
121°C. The contents of the autoclave are maintained at this temperature for 15-20
minutes. Explain why these conditions are necessary for effective sterilisation. (2)
(b) Suggest why the cotton wool plugs of the flasks are covered with aluminium foil. (1)
(c) Describe and explain two precautions, other than the use of sterile equipment, you
would need to take to prevent contamination of cultures of microorganisms. (4)
(Total 7 marks)

SAQ 31 A suspension of bacteria was spread evenly over solid medium in a petri dish. The culture
was incubated at 25 °C for 12 hours.
After 12 hours two discs were placed on the surface of the medium. Each disc had been
soaked in a different antibiotic (antibiotic A and antibiotic B).
The culture was then incubated for 48 hours to allow the antibiotics to diffuse into the medium.
The appearance of the culture before and after incubating with the antibiotic-soaked discs is
shown below.
Antibiotic-
soaked disc

B B

A A
Bacterial
colonies

Appearence of culture after 12 hours, Appearence of culture 48 hours

on adding the antibiotic-soaked discs after adding the discs
Compiled by Stafford Valentine Redden 34
 (a) Describe the effect of each antibiotic on the growth of bacteria. Suggest a reason for the
effect of each antibiotic.
Antibiotic A
Antibiotic B (4)
(b) Suggest why antibiotics such as penicillin affect bacterial cells but not the cells of the
patient they are used to treat. (3) (Total 7 marks)

Reliability: When the variability in replicated results is very high, the reliability is relatively low. This may
be due to certain variables not being controlled of faulty experimental procedures. Range bars or error
bars on the graph will give a fair indication of the reliability. If there is considerable overlap between error
bars, then variability is very high and reliability is low. Students should be able to identify factors that may
decrease the reliability of the results.

Accuracy: accuracy can be defined as the difference between the actual values and the measured
values. If the difference is high, then accuracy is low and vice versa. Accuracy can be improved by using
appropriate apparatus and methods for making measurements.

Question one (Core practical) Data organisation.

a. Tabulation
The Independent variable comes in the first column. Arrange values in ascending order.
The independent variable is controlled by the experimenter.
The dependent variable is measured to give the results.
The dependent factor depends on the independent variable.

Label all columns and rows appropriately and accurately.

Include SI units in the headings of the columns and rows.
Be consistent with significant figures / decimal places.
You are generally asked to find the mean or the percentage difference.
Mean = sum of readings / number of readings. ( e.g. Mean = 12 + 11.2 + 13.1 / 3 = 12.1)

Percentage Difference = (Difference / Initial reading) x 100

b. Graph
Axes: Independent variable on X - axis. Label appropriately with units.
Dependent variable on Y - axis. Label appropriately with units.
Scale: The curve should cover more than 50 % of the graph paper.
Plot all points accurately. Mark the plotted point with a dot and a circle or with a cross.
Line: Join each point with a neat straight line, passing exactly through the point. Do not extrapolate.
c. Describe the trends in the graph. Do NOT make theoretical assumptions. Make conclusions based on the
experimental data. Refer to the hypothesis being investigated, while making conclusions.
d. Limitations are genuine sources of error. Look out for variables like temperature, pH, etc. not being
controlled. Also check if the experiment has been replicated.

Every experiment has two main components:

1. Control of variable / factors.

Ensure that you control all Variables and factors that can affect your results. State sensible values, with SI units
and describe sensible ways of controlling these variables. Describe the type of apparatus that could be used.

Controlling factors effectively will improve the reliability of your results.

2. Making quantifiable measurements.
The results of your experiment should have numerical values, so that the data can be analysed and results
can be drawn.
Using an appropriate method and suitable apparatus can improve the accuracy of
your results.

Note: Examiners usually check whether you know the factors that need to be controlled for the
core practical. They also expect you to devise or be aware of suitable methods of making accurate
measurements.
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