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Year 12 As Core Practicals
Year 12 As Core Practicals
).
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.
Mineral ion
Nitrate
Potassium
Magnesium
Calcium
Concentration of ions in nutrient solution / arbitrary units
At start of experiment At end of experiment
7.0
3.0
2.0
5.0
1.8
0.0
2.1
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 30
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.
Week
Dry mass / mg
Lemna polyrrhiza Lemna gibba
0
1
2
3
4
5
6
7
8
25
69
87
106
87
100
87
81
37
22
84
169
197
289
344
344
347
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
Lemna polyrrhiza
Experiment C
Lemna polyrrhiza
0
1
2
3
4
5
6
7
8
63
100
188
306
381
448
562
594
641
50
119
200
259
291
356
350
369
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? (1)
(ii) Suggest an explanation for this difference. (2) (Total 15 marks)
Compiled by Stafford Valentine Redden 31
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 C for 15 minutes.
Procedure for pouring a plate
Allow liquid agar to cool to about 50 C (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 cm
3
) 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 C, 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 32
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
C. At this temperature, the agar will be cool enough to handle safely, but will remain molten.
Agar starts to set below about 42 C.
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 C 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 10
7
bacteria and incubated for three days.
Compiled by Stafford Valentine Redden 33
The results are shown in the table below.
0
10
20
30
40
50
100
200
2
3
6
7
11
14
24
40
1
4
9
14
23
34
7
0
2
4
3
5
2
1
3
6
Number of colonies
Compound A Compound B Compound C
Concentration of compound
/ cm g
3
(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 g 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.
E
D
A
B
C
Clear
zones
Bacteria growing
on agar
Antibiotics discs
(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 34
(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.
Heat Heat
Left to cool
Water
Steam
Mass
added
Valve
closed
Valve
open
Aluminium
foil
Cotton
wool plug
(a) The pressure of steam builds up to 103 kPa. This raises the boiling point of water to
121C. 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.
A A
B B
Antibiotic-
soaked disc
Bacterial
colonies
Appearence of culture after 12 hours,
on adding the antibiotic-soaked discs
Appearence of culture 48 hours
after adding the discs
Compiled by Stafford Valentine Redden 35
(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.