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IBO2012
SINGAPORE
CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

Country: _____________________

Student Code: ________________

23rd INTERNATIONAL BIOLOGY OLYMPIAD

8th 15th July, 2012
SINGAPORE

PRACTICAL TEST 1
CELL & MOLECULAR BIOLOGY
Total points: 100

Duration: 90 minutes

Page 1 of 9

IBO2012
SINGAPORE
CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

Dear Participants

In this test, you have been given the following task:

Task: Gene mapping by restriction endonuclease digestion of DNA fragments
Part A. Confirmation of insertion of human DNA in a cloning plasmid. (80 points)
Part B. Determination of orientation by which the fragment is inserted. (20 points)

Use the Answer Sheet, which is provided separately, to answer all the questions.

The answers written in the Question Paper will NOT be evaluated.

Write your answers legibly in ink.

Please make sure that you have received all the materials and equipment listed for each task.
If any of these items are missing, please raise your hand immediately.

Stop answering and put down your pen IMMEDIATELY when the bell rings.

At the end of the test, place the Answer Sheet and Question paper in the envelope provided.
Our Assistants will collect the envelope from you.

Have fun and Good Luck!

Page 2 of 9

IBO2012
SINGAPORE
CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

Materials and equipment:

Materials and equipment

Quantity

Unit

restriction endonuclease RE1 (Ndel) (kept on ice)

4 l

tube

restriction endonuclease RE2 (EcoRl) (kept on ice)

4 l

tube

10 l x 4

tube

miliQ water (labelled W)

tube

DNA electrophoresis gel tank and power supply

set

micropipettes and tips in boxes (p10, p100)

piece

stopwatch

piece

DNA ladder (as internal size markers, L1 for 100 bp range and
L2 for 1 kbp range) (on ice)

tube

DNA loading dye (blue in colour)

tube

pre-cast gel in holder (already placed in running buffer)

piece

large petri dish (for placing the gel for imaging purposes)

piece

card with your country code (in a clip holder): for signalling for
assistance

piece

floating rack (labelled with your country code)

piece

micro-centrifuge

set

water-bath 37 C (there is one assigned for your usage)

set

gel doc (there is one assigned for your usage)

set

DNA test samples in enzyme buffer (labelled T) (on ice)

Page 3 of 9

IBO2012
SINGAPORE
CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

Task (100 points)

Gene mapping by restriction endonuclease digestion of DNA fragments
Introduction
Genetic mapping is routinely used in analysing the order and the identities of DNA fragments. This
technique is based on the unique profiles of DNA fragments generated after DNA digests with
specific combination of restriction endonucleases (RE) and revealed by DNA gel electrophoresis. It
is extremely powerful for gene cloning, studying gene function and regulation, for finding candidate
genes for diseases and their diagnosis and also as a forensic tool.

Part A. Confirmation of insertion of human DNA in a cloning plasmid. (80 points)

Using this technique, you are now tasked to confirm that a fragment of human DNA X
(approximate size: 760 base pairs) has been inserted into a cloning plasmid or vector V (circular
and approximate size: 2570 base pairs). You are required to design and carry out DNA digests by
incubating DNA T with the restriction endonucleases by following the general protocol of
incubation and electrophoresis given (details described below). After the gel electrophoresis, your
results will be revealed by DNA staining (this will be performed by lab technicians), analysed and
data interpreted.

Protocol and Procedures

1.

Design your DNA digests (you may do a maximum of 4 tubes) in a total volume of 20 l by
using the Table in the Answer Sheet.
Q1.1

(20 points) Record the desired amounts of reagents in your plan. One example is
already given for Tube 2 in the table provided. All units are in l.

Page 4 of 9

IBO2012
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CELL & MOLECULAR BIOLOGY

2.

PRACTICAL TEST 1

Prepare the mixtures by carefully pipetting the correct amount of the reagents and gently mix
them by pipetting them up and down in each tube. Label the tubes. Do not contaminate one
sample with another when preparing the mixture. Use a clean pipette tip for each operation.
Note: use p10 micropipette (white-coded) for pipetting reagents of less than 10 l. [NOTE:
there will be a penalty of 20 points if additional samples are requested. Please prepare the
samples carefully.]

3.

Spin down the mixture by placing all four tubes in the micro-centrifuge (please balance the spin
by placing tubes opposite to each other). During preparation and after spinning, always keep
the tubes on ice.

4.

After all the tubes have been prepared, remove them from the ice and place them into the
labelled floatation rack and incubate them for 20 minutes (stopwatch is provided) at 37 C in the
water bath assigned to you. Make sure that you retrieve your own samples after the 20 minutes
incubation time.

5.

During this 20 minute incubation duration, answer the following questions in the Answer Sheet:
Q1.2

(2 points 5 = 10 points) Indicate true statement(s) with a tick () and false

statement(s) with a cross ().
a.

Each RE cuts DNA at a specific sequence.

b.

Each RE cuts DNA only at the 3 and 5ends.

c.

RE are most effective in digesting DNA at 4 C.

d.

RE can be kept at room temperature for months.

e.

Unlike exonucleases, RE only cuts DNA internally.

Page 5 of 9

IBO2012
SINGAPORE
CELL & MOLECULAR BIOLOGY

Q1.3

PRACTICAL TEST 1

(2 points 5 = 10 points) Which of the following principles is true of separating DNA

by gel electrophoresis? Indicate true statement(s) with a tick () and false statement(s)
with a cross ().
a.

DNA fragments are overall positively charged.

b.

The smaller DNA fragments move faster across the gel under the electric current.

c.

The smaller DNA fragments are lesser charged than the larger fragments hence
they move faster across the gel.

d.

The relative density of the gel matrix affects how long the separation takes.

e.

The voltage applied to the electrophoresis is determined by how much DNA is

loaded in the gel.

6.

When the 20 minutes of DNA digests duration is up, retrieve your own tubes from the water
bath.

7.

Add 4 l of DNA loading-dye (blue colour). Mix them well by pipetting the mixture up and down
and spin down any residual liquid using the micro-centrifuge.

8.

Using the p100 micropipette (yellow-coded), load 15 l of the sample mixture of DNA digests
with the loading dye into the wells of the agarose gel provided. Make sure that you position
the pipette tips carefully on top of the wells and gently deliver the mixture to the wells without
spilling them. Load 15 l of each of the Markers, L1 and L2. Add your samples according to
the following scheme of lanes, starting from the left end of the gel.
Marker L1

9.

Tube 1

Tube 2

Tube 3

Tube 4

Marker L2

Cover the gel with a lid and connect the power supply to run at 100 volts for 20 min. Please be
careful and do not touch any part of the electrodes and power supply.

Page 6 of 9

IBO2012
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CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

10. Check regularly that the samples have entered the wells and are indeed running towards the
positive electrode. If you need help from the technicians to ensure proper runs for the samples,
please signal for assistance by clipping your signal card at the edge of right wall of your cubicle.

11. While waiting for the gel run, answer the following questions in the Answer Sheet:

Q1.4

(20 points) Consider the following scenario: A piece of linear human DNA (1 kbp) was
digested by a particular enzyme RE3, resulting in 2 fragments of 650 bp and 350 bp.
The same piece of 1 kbp DNA was digested with another enzyme RE4, releasing 2
fragments of 800 bp and 200 bp. And when this 1 kbp DNA was digested with RE3 and
RE4 together, 3 fragments of DNA were generated, 650 bp, 200 bp and 150 bp.

Sketch a linear map of this piece of DNA by indicating the position of RE3 and RE4
digests in the space provided. An example of such a sketch is provided below as a
guide.

12. When the 20 minutes of gel running time is up, turn off the power supply and remove the lid of
the gel tank. Carefully remove the gel (still on the gel tray) and place it on the petri dish
provided. Bring your gel to the gel doc that has been assigned for your usage and the
technician will photograph it for you.

13. Bring your gel and the photograph back to your cubicle and use your signal card to get
assistance for an invigilator to staple it in the space provided on the Answer Sheet.
Q1.5

(10 points) Your skills in running a gel will be assessed by the quality of the gel
produced.
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CELL & MOLECULAR BIOLOGY

PRACTICAL TEST 1

14. Based on the gel results, answer the following questions in the Answer Sheet:
Q1.6

(1 point 5 = 5 points) Using the DNA ladder markers (in basepairs) provided below
as the reference, estimate the sizes of the fragments/bands. You may draw a line
across the band of your query and the size marker to do the estimation. How many
fragment(s) of DNA were generated by RE1 and RE2? And what is/are the estimated
size(s)? Answer using numerals.

L1: 100 bp DNA Ladder

Q1.7

L2: 1 kb DNA Ladder

(1 point) What is the estimated size of the test DNA sample (T)? Answer using
numerals.

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IBO2012
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CELL & MOLECULAR BIOLOGY

Q1.8

PRACTICAL TEST 1

(1 point) Based on your results, is the test DNA sample (T) larger, smaller or the same
size as the empty vector? Indicate your answer with a tick () in the correct box.

Q1.9

(1 point) Does the test DNA sample (T) contain any insert? Indicate yes with a tick ()
and no with a cross ().

Q1.10 (2 points) Uncut DNA appears to move faster than any of the samples digested with
RE2. Why? Indicate your answer with a tick () in the correct box.
a.

The smaller fragment size of uncut DNA is due to DNA degradation.

b.

The uncut DNA is more compact and therefore moves faster through the gel.

c.

RE2 still binds to the DNA and therefore slows down their movement through gel.

Part B. Determination of orientation by which fragment was inserted. (20 points)

Q1.11 (20 points) Construct possible restriction map(s) for the DNA T by indicating the relative
position of RE1 and RE2 and the distance between them in the Answer Sheet. An example
of such a map is provided below as a guide.

END OF PAPER

Page 9 of 9

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Country: _____________________

Student Code: ________________

23rd INTERNATIONAL BIOLOGY OLYMPIAD

8th 15th July, 2012
SINGAPORE

PRACTICAL TEST 2
MICROBIOLOGY & BIOCHEMISTRY
Total points: 100

Duration: 90 minutes

Page 1 of 13

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Dear Participants

In this test, you have been given the following two tasks:
Task 1:

Bacteriophage: an effective agent in the killing of pathogenic bacteria. (50 points)

Part A: Effects of Phage and antibiotics on the killing of antibiotic-resistant E. coli (31
points)
Part B: Phage titre and multiplicity of infection (19 points)

Task 2:

Titration of an amino acid. (50 points)

Use the Answer Sheet, which is provided separately, to answer all the questions.

The answers written in the Question Paper will NOT be evaluated.

Write your answers legibly in ink.

Please make sure that you have received all the materials and equipment listed for each task.
If any of these items are missing, please raise your hand immediately.

Stop answering and put down your pen IMMEDIATELY when the bell rings.

At the end of the test, place the Answer Sheets and Question paper in the envelope provided.
Our Assistants will collect the envelope from you.

Have fun and Good Luck!

Page 2 of 13

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Materials and equipment:

For Task I: Bacteriophage: an effective agent in the killing of bacteria
Materials and equipment

Quantity

Unit

micropipette tips 10l

box

micropipette tips 200l

box

micropipette tips 1000l

box

micropipette 1 - 10l

piece

micropipette 2 - 20l

piece

micropipette 20 - 200l

piece

micropipette 100 - 1000l

piece

microfuge tube rack

piece

cuvette rack

piece

many

tube

stock E. coli culture (1 107 cells/ml) in LB broth

tube

LB broth (in a 50 ml Falcon tube)

tube

sterile deionized water in microfuge tube

tube

ampicillin stock (1 mg/ml) dissolved in deionized water

tube

bacteriophage stock (108 pfu/ml) in deionized water

tube

cuvettes (in a beaker)

piece

stopwatch

piece

floating rack (labelled with your country code)

piece

water-bath 37 C (there is one assigned for your usage)

set

UV-VIS Spectrophotometer (there is one assigned for your usage)

set

photographs of E. coli plates (A to H)

set

Quantity

Unit

25 ml burette

piece

25 ml pipette

piece

100 ml beakers

piece

magnetic stirring bar

piece

magnetic stirrer

set

microfuge tubes (in a beaker)

For Task II: Titration of an amino acid

Materials and equipment

Page 3 of 13

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

pH meter with electrode

set

pipette bulb

piece

Kimwipe papers

box

retort stand with clamps

set

0.3024 M standardized NaOH solution

100

ml

Amino acid Z solution of unknown concentration

80

ml

Page 4 of 13

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Task I (50 points)

Bacteriophage: an effective agent in the killing of bacteria
Part A. Effects of Phage and antibiotics on the killing of antibiotic-resistant E. coli (31 points)
Introduction
A bacteriophage is a virus that infects bacterial cells. Certain bacteriophages can kill bacteria cells
by lysis. The bacteriophage is now recognized as an effective agent in the killing of pathogenic
bacteria. This provides a good alternative to antibiotics in our combat against disease-causing
bacteria that might be resistant to traditional antibiotics. You are required to design a simple
experiment, with proper controls, to examine the killing efficiency of phage of an ampicillin-resistant
E. coli. Answer the following questions in the Answer Sheet and follow the instructions given
below.

Q1.1

(1 point) To dilute the E. coli culture from 1 107 cells/ml to 2 105 cells/ml, what would be
the dilution factor needed?

Q1.2

(1 point) For 1 ml of E coli culture at a cell density of 2 105 cells/ml, the final concentration
of ampicillin used should be 10 g/ml. What would be the volume of ampicillin stock (1mg/ml)
used?

Q1.3

(1 point) For 1 ml of E coli culture at a cell density of 2 105 cells/ml, final titre of phage
used should be 106 pfu/ml. What would be the volume of phage stock (108pfu/ml) used?

Q1.4

(1 point 15 = 15 points) With the above calculated dilution factors, fill in the table in the
Answer Sheet with your experimental plan. One example is already given for Tube 1 in the
table provided. All units are in l. Carry out your proposed experiment by incubating the
four tubes (placed in the labelled floating rack) for 40 minutes (stop watch provided) in the 37

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IBO2012
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MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

C water bath assigned to you. Hand over the floating rack to the technician at the water
bath.

After incubation, transfer your samples to the cuvettes labelled 1 to 4. In order to observe
the killing of bacteria cells, measure the absorbance at 595 nm wavelength. Bring your
samples to the spectrophotometer that is allocated for your use and hand over your samples
to the technician. You are to record your own readings as the samples are measured.

Q1.5

(0.75 2 + 1.5 points 6 = 10.5 points) Fill in the absorbance reading at 595 nm of the
different tubes of reactions in the table provided in the Answer Sheet. Taking 1 absorbance
unit of the E. coli cells at 595 nm to be equivalent to 1 107 cells/ml, what are the cell
densities of the E. coli in the respective reaction tubes?

Q1.6

(0.5 points 5 = 2.5 points) Which of the following are correct? Indicate correct answer(s)
with a tick () and incorrect answer(s) with a cross ().
a.

Due to the ampicillin resistance, the bacteria cell wall prevented easy penetration of the
antibiotics, but allowed the phages to enter the E. coli cells to cause lysis.

b.

The ampicillin resistance in the E. coli did not prevent the ability of the phage to adsorp
onto the bacteria cells.

c.

The bacteriophage likely has a lytic life cycle of around 20 to 30 mins and hence lysis of
the E. coli was observable during the short experiment.

d.

The temperature of 37 C was not the correct temperature for ampicillin to kill the E. coli
cells.

e.

The phages competed with the E. coli for the nutrients in the LB broth and the bacteria
cells lysed due to insufficient nutrients.

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MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Part B. Phage titre and multiplicity of infection (19 points)

The Table below shows the legends for photographs of E. coli lawns that are untreated and infected
with bacteriophages. The E. coli culture used had a starting cell density of 0.5 104 cells/ml. 0.5 ml
of phage was used to infect the E. coli cells. Serial dilutions of the phage culture were made as
indicated and used for infection. (The photographs labelled A to H will be provided as part of
materials for the lab task).

-6

B = 10 dilution

-4

D = 10 dilution

E = 10 dilution

-2

F = 10 dilution

G = neat phage

H = E. coli lawn
uninfected by phage

A = 10 dilution

C = 10 dilution

-5

-3

-1

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IBO2012
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MICROBIOLOGY & BIOCHEMISTRY

Q1.7

PRACTICAL TEST 2

(2 points 4 = 8 points) Based on the number of plaques observed in the photos, calculate
the number of plaques that would be observed if the original undiluted phage culture were
used.

Q1.8

(3 points) To estimate the titre of a phage culture, serial dilutions as shown in the photos (A
to H) are normally performed. Based on the number of plaques shown, indicate with a tick ()
which is the best dilution to confirm the phage titre.

Q1.9

(4 points 2 = 8 points) Using the information given and your answers above, determine:
a.

the plaque forming units per milliliter (pfu/ml) of the phage culture used and

b.

the multiplicity of infection(defined as the ratio of phages to E. coli),

at the best dilution determined in Q1.8.

Page 8 of 13

IBO2012
SINGAPORE
MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

Task II (50 points)

Titration of an Amino Acid
Introduction
Amino acids are organic molecules possessing both carboxyl and amino groups. Table 1 shows the
20 amino acids that cells use to build their thousands of proteins. The majority of the standard
amino acids are diprotic molecules since they have two dissociable protons: one on the amino
group and the other on the carboxyl group; there is no dissociable proton in the R group.
Recall: For an acid HA, the acid dissociation constant for the equilibrium of HA

H+ + A is Ka.

Ka = [H+][A] / [HA]

More often, the strength of acids is expressed in terms of the pKa of the acid:
pKa = log Ka

In the titration of such a diprotic amino acid, the titration will thus occur in two steps as the more
acidic carboxyl group (lower pKa1) and the less acidic amino group (pKa2) successively lose their
protons.

In addition, the pH at which the net charge on the molecule is zero is called the isoelectric point (pI)
of the molecule, a useful constant in characterizing and purifying molecules. Using a titration curve,
the pI can be empirically determined as the inflection point between the pKas of the anionic and
cationic forms.

Page 9 of 13

IBO2012
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MICROBIOLOGY & BIOCHEMISTRY

PRACTICAL TEST 2

The apparent pKa values for the two dissociation steps may be extrapolated from the midpoints of
each step. This can be shown by the Henderson-Hasselbalch equation:
pH = pKa + log { [A] / [HA] }
The pKa1 (pKa for the carboxyl acid group) is where half the acid group has been titrated. Therefore
the equation becomes:
pH = pKa
Similarly, the pKa2 (pKa for the amino group) can be determined.
In this experiment, you will titrate an unknown amino acid Z and determine its pI, pKa1 and pKa2.

Procedure
1.

Fill the burette with the standardized NaOH solution. Record the exact concentration of this
standardized NaOH solution in the Answer Sheet.

2.

Pipette 25 ml of the unknown amino acid solution Z into a clean 100 ml beaker.

3.

Carefully place the pH probe and a magnetic stirring bar into the amino acid solution, so that
the probe is far enough into the solution, but not touching the stirring bar or beaker. Clamp and
adjust the pH probe such that the stirring bar will not hit the probe while stirring. DO NOT
TOUCH THE CALIBRATION.

4.

Titration 1
Rinse the pH probe with deionized water. Dry the probe gently with a piece of Kimwipe paper.
Determine the pH of the amino acid solution Z before the addition of NaOH. Next, titrate the
amino acid solution with the NaOH from the burette. Add approximately 1.00 ml of the NaOH to
the amino acid at a time. Record the exact volume dispensed and the pH of the solution after
every 1.00 ml interval in the Answer Sheet. Continue until approximately 25 ml of NaOH has
been added.

5.

Repeat the titration (Titration 2)

Rinse the pH probe with deionized water. Dry the probe gently with a piece of Kimwipe paper.
Refill the burette with the standardized NaOH solution and repeat steps 2 4.
Page 10 of 13

IBO2012
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MICROBIOLOGY & BIOCHEMISTRY

Q2.1

PRACTICAL TEST 2

(3 points 3 = 9 points) Table 1 shows the chemical structures of the twenty standard
amino acids. With reference to these structures, draw structures to show the complete
dissociation of glycine, proline and asparagine.

Q2.2

(3 points 2 = 6 points) For both titrations, record the volume of NaOH (ml) added during
the titration and the observed pH value for the unknown amino acid.

Q2.3

(5 points 2 = 10 points) Using your data, plot the graphs of each titration run (pH versus
Vol. of NaOH (ml)) in Graphs 1 and 2 provided in the Answer Sheet.

Q2.4

(2 points 2 = 4 points) From your titration curves, find the pI and label it on each graph.
Q2.4.1 (2 points) What is the mean pI?

Q2.5

(4 points 2 = 8 points) Find and label the pKa1 and pKa2 on each graph.
Q2.5.1 (2 points 2 = 4 points) What is the mean pKa1 and pKa2?

Q2.6

(5 points) 0.9210 g of the unknown amino acid Z was dissolved in 80 ml of deionized water.
Determine the molecular weight of the unknown amino acid Z. Note: In order to start with a
fully protonated amino acid, HCl solution has been added. This is equivalent to 3.2 ml of the
NaOH solution. To determine the actual number of moles of NaOH needed to reach the pl,
subtract 3.2 ml from the volume of NaOH used to reach the first end point.

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MICROBIOLOGY & BIOCHEMISTRY

Q2.7

PRACTICAL TEST 2

(2 points) Based on Table 2, identify amino acid Z.

a.

glycine

b.

proline

c.

asparagine

d.

tyrosine

e.

tryptophan

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PRACTICAL TEST 2

Table 1. Structures of the 20 standard amino acids.

Table 2. Molecular weights of amino acids

Amino acid

MW (g/mole)

Glycine

75

Proline

115

Asparagine

132

Tyrosine

181

Tryptophan

204

END OF PAPER
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IBO2012
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PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

Country: _____________________

PRACTICAL TEST 3

Student Code: ________________

23rd INTERNATIONAL BIOLOGY OLYMPIAD

8th 15th July, 2012
SINGAPORE

PRACTICAL TEST 3
PLANT DIVERSITY, ANATOMY & PHYSIOLOGY
Total points: 100

Duration: 90 minutes

Page 1 of 14

IBO2012
SINGAPORE
PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

Dear Participants

In this test, you have been given the following two tasks:
Task I: Plant diversity and anatomy. (60 points)
Part A: Morphology of seedlings (14.25 points)
Part B: Seed morphology and anatomy (27.25 points)
Part C: Ficus propagule dissection (5 points)
Part D: Functional, ecological and phylogenetic aspects of seeds and seedlings (13.5
points)
Task II: Plant anatomy and physiology. (40 points)
Part A: Anatomy of a plant stem (13 points)
Part B: Study of leaf epidermis and physiology (15 points)
Part C: Interpretation of photosynthetic data (12 points)

Use the Answer Sheet, which is provided separately, to answer all the questions.

The answers written in the Question Paper will NOT be evaluated.

Write your answers legibly in ink (you may use a pencil for diagrams).

Please make sure that you have received all the materials and equipment listed for each task.
If any of these items are missing, please raise your hand immediately.

Stop answering and put down your pen IMMEDIATELY when the bell rings.

At the end of the test, place the Answer Sheets and Question paper in the envelope provided.
Our Assistants will collect the envelope from you.

Have fun and Good Luck!

Page 2 of 14

IBO2012
SINGAPORE
PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

Materials and equipment:

For Task I: Plant diversity and anatomy
Materials and equipment

Quantity

Unit

Seedlings: A, B, C and D (in plastic cups)

specimen

Seeds/propagules: 1 to 7 (in labelled plastic bags)

specimen

Specimen E (in labelled plastic bag)

specimen

razor blade (use ONLY for Seed 3 and Seed 5)

piece

Scissors (use for Seed 4 and Specimen E)

pair

hand lens

piece

Materials and equipment

Quantity

Unit

Leaves, L (in petri dish L)

piece

Stems, S (in petri dish S)

piece

concentrated HCl (in bottle labelled H, placed within a beaker)

bottle

Phloroglucin stain (in bottle labelled P)

bottle

water (in wash bottle)

bottle

filter paper

sheet

forceps

pair

razor blade

piece

plastic dropper

piece

petri dish (with water, labelled W)

piece

petri dish (labelled LL, LU and SS with the correct student code)

piece

beaker (small)

piece

glass slides

piece

cover slips

piece

compound microscope

set

For Task II: Plant anatomy and physiology

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IBO2012
SINGAPORE
PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

Task I (60 points)

Plant diversity and anatomy
Part A. Morphology of seedlings (14.25 points)
Q1.1

(0.5 points 20 = 10 points; 2 points for quality of drawing; 2.25 points for not
damaging specimens) Make a simple schematic diagram of each seedling (A D) in the
corresponding space provided in the Answer Sheet and label the following (if present) with
a to e and indicate if any are absent:
a.

cotyledons

b.

epicotyl

c.

hypocotyl

d.

leaves

e.

seed coat

Part B. Seed morphology and anatomy (27.25 points)

Each seedling (A to D) from Part A comes from seeds 1-4 respectively.
Q1.2

(0.25 points 11 = 2.75 points) Draw each whole seed (1 6) in the corresponding space
provided in the Answer Sheet (Note: (1) you do not need to draw Seed 7; (2) seed coat of
Seed 5 has been removed). Label the following (if present) with a and b and indicate if any
part is absent:
a.

seed coat

b.

site of attachment of funiculus

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PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

Q1.3

PRACTICAL TEST 3

(0.5 points 24 = 12 points; 1 point for quality of drawing) After drawing the exterior of
each seed, dissect in longitudinal section and draw the sectioned seed in the corresponding
space provided in the Answer Sheet. (Note: Some seeds have been pre-cut for your
convenience. The seed coat of Seed 5 has been removed.). Label/indicate the following
items on your diagram with a to d:

Q1.4

a.

cotyledons

b.

food storage

c.

hypocotyl

d.

seed coat

(0.5 points 23 = 11.5 points) Indicate the likely ploidy (1N, 2N or 3N) of each of the items
labelled a to d in Q1.3 in the Answer Sheet.

Part C. Ficus propagule (dispersal unit) (5 points)

Q1.5

(1 + 1 + 3 points) Dissect Specimen E longitudinally and draw the longitudinal section of the
propagule (dispersal unit). Draw and label an enlarged section to show in detail the features
of a to c.
a.

fruit

b.

seed

c.

stigma

Part D. Functional, ecological and phylogenetic aspects of seeds and seedlings (13.5 points)
Q1.6

(0.5 points 9 = 4.5 points) Based on the observations in Parts A, B and C, and the
information given in the Table provided in the Answer Sheet, indicate the primary function
of the cotyledon with P (for photosynthesis) or S (for storage of nutrients) and the probable

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PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

germination pattern with O (orthodox: seed can undergo dormancy) or R (recalcitrant: seed
does not undergo dormancy).

Q1.7

(1 point 5 = 5 points) Based on the information in the Table and the diagrams that you
have drawn, determine if each of the following statements is true () or false ().
a.

All tropical plants have recalcitrant seeds.

b.

Gymnosperms have at most two cotyledons.

c.

Cotyledon function is phylogenetically constrained within plant families.

d.

Seed size varies considerably in plant families and is probably not phylogenetically
constrained.

e.

Q1.8

Large seed size may be advantageous for some tropical rainforest plants.

(1 point 4 = 4 points) The phylogenies of Malvaceae and Moraceae, and a simplified

phylogeny of seed plants are provided on the next page. Using this information, as well as
the specimens that you have observed today, determine if each of the following statements
is true () or false (), or if there is insufficient evidence to conclude () in the Answer
Sheet.

a.

Large seeds have evolved independently in multiple lineages.

b.

Large seeds have evolved on more than one occasion in some lineages.

c.

Recalcitrant seeds are more associated with tropical plants than with temperate plants.

d.

Gymnosperms are unable to produce fleshy structures associated with animal dispersal
because they lack ovaries.

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PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

Phylogeny of Malvaceae

Phylogeny of Moraceae

Simplified phylogeny of seed plants

Gymnosperms
(Seeds 5, 6, 7)

Monocots

Moraceae
+ related families
(Seed 2, Specimen E)

Malvaceae, Sapindaceae
+ related families
(Seeds 1, 3, 4)

Asteraceae + related families

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PLANT DIVERSITY, ANATOMY & PHYSIOLOGY

PRACTICAL TEST 3

Task II (40 points)

Plant anatomy and physiology
Part A. Anatomy of a plant stem (13 points)
Procedure:
1.

Using the razor blade, cut as thin as possible transverse sections of the stem, S.

2.

Float the cross-sections in water in petri dish, W.

3.

Place a drop of water onto a glass slide and transfer the best cross-section to the centre of the
slide.

4.

Place a small drop of phloroglucin stain (P) onto the cross-section, followed by a small drop of
concentrated hydrochloric acid, HCl (H). Be very careful when handling H as it is corrosive.

5.

Leave for 1 minute and then remove excess stain with the filter paper.

6.

Cover the cross-section with a coverslip and examine it using the compound microscope under
low magnification (4 objective lens).

Answer the following questions in the Answer Sheet:

Q2.1

(1 point) Is S a monocot stem or dicot stem? Indicate M for monocot and D for dicot.

Q2.2

(1 point) Is there any pith? Indicate presence of pith with a tick () and absence with a
cross ().

Q2.3

(1 point) Where is the vascular bundle in the stem located? Indicate centre with C and
periphery with P.

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PRACTICAL TEST 3

Q2.4 (0.5 points 3 = 1.5 points) Based on your observations, is S the stem of a shrub, tree,
or herb? Indicate correct answer(s) with a tick () and incorrect answer(s) with a cross
() in the Answer Sheet.

Q2.5

(0.5 points) Identify the tissue (a e) that is stained red. Indicate the correct answer
with a tick () in the Answer Sheet.

7.

a.

cortex

b.

endodermis

c.

epidermis

d.

phloem

e.

xylem

When you have completed Part A, place your slide with the stem section into the petri dish SS
for grading purposes.
Q2.6

(8 points)

Part B. Study of leaf epidermis and physiology (15 points)

(i)

Lower epidermis

Procedure:
1.

Peel off the lower epidermis of the leaf, L, with a pair of forceps.

2.

Place it in a drop of water on a glass slide and cover the peeled layer with a cover slip.

3.

Examine it using the compound microscope under10 objective lens.

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PRACTICAL TEST 3

Answer the following questions in the Answer Sheet:

Q2.7

(2 points) Do you see any stomata? Indicate presence of stomata with a tick ()
and absence with a cross ().

Q2.8

(3 points) Measure the lengths and widths of FIVE (5) epidermal cells that are
representative of the majority of the cells. Smallest unit in the eyepiece micrometer
as seen under 10 objective lens is 10 m. Calculate the mean values and fill in your
answer in the table provided.

4.

When you have completed Part B (i), place your slide with the epidermal peel into the
petri dish LL for checking purposes. The slide will also be used to check for accuracy of
your measurement of epidermal cell dimensions.

(ii) Upper epidermis

Procedure:
1.

Peel off the upper epidermis of the leaf, L, with a pair of forceps. You may either use
the same leaf as before or a fresh leaf from petri dish L.

2.

Place it in a drop of water on a glass slide and cover the peeled layer with a cover slip.

3.

Examine it using the compound microscope under 10 objective lens.

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PRACTICAL TEST 3

Answer the following questions in the Answer Sheet:

Q2.9

(2 points) Do you see any stomata? Indicate presence of stomata with a tick ()
and absence with a cross ().

Q2.10 (3 points) Measure the lengths and widths of FIVE (5) epidermal cells that are
representative of the majority of the cells. Smallest unit in the eyepiece micrometer
as seen under 10 objective lens is 10 m. Calculate the mean values and fill in your
answers in the table provided.

4.

When you have completed Part B (ii), place your slide with the epidermal peel into
the petri dish LU for checking purposes. The slide will also be used to check for
accuracy of your measurement of epidermal cell dimensions.

Q2.11 (0.5 point 3 = 1.5 points) Based on your observations in Part B (i) and (ii), indicate
the correct answer(s) with a tick () and incorrect answer(s) with a cross ().
a.

There are more stomata in the lower epidermis than in the upper epidermis.

b.

Epidermal cells of the upper epidermis are smaller than those of the lower
epidermis.

c.

Stomata are separated from each other by at least one cell.

Q2.12 (1 point) Based on your observations, determine what type of plant this is. Indicate
the correct answer with a tick () in the Answer Sheet.
a. hydrophyte
b. mesophyte
c. xerophyte

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PRACTICAL TEST 3

Q2.13 (0.5 point 5 = 2.5 points) Some statements about stomatal structure, function and
development are given below. Indicate true statement(s) with a tick () and false
statement(s) with a cross ().
a. Stomata consist of a pair of highly specialized guard cells that are usually
surrounded by a pair of larger subsidiary cells.
b. Guard cells differ significantly from other epidermal cells in that they have
chloroplasts.
c. Chloroplasts of guard cells differ from mesophyll chloroplasts in that they lack
grana.
d. The number of stomata on any leaf surface is under genetic control and is not
modified by any environmental factors.
e. Stomatal development involves asymmetric cell divisions.

Part C. Interpretation of photosynthetic data from plants measured at different CO2 concentrations
(12 points)

Introduction
Single leaves from plants A and B that had been grown under full sunlight in the same greenhouse
were studied. The responses of their net photosynthetic CO2 assimilation rates to varying levels of
ambient CO2 under saturating light intensity of 1,200 mol quanta m2 s1 at 25 C and 21% O2
measured on leaves in the laboratory are given on the next page:

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PRACTICAL TEST 3

Net photosynthetic CO2 assimilation rate

-1

Ambient CO2 concentration (l l )

(mol CO2 m2 s1)
Plant A

Plant B

20

0.5

-4

40

11

-1

60

19

2.5

80

28

5.5

100

33

180

41

18

300

44

27

400

44

32

600

44

40

800

44

44

1000

44

45.5

Q2.14 (4 points). Plot a graph by using the data above for Plant A and B in Graph 1 provided in
the Answer Sheet. Use an X-axis scale from 0 to 1000 l l-1.

Based on Graph 1, answer the following questions in the Answer Sheet:

Q2.15 (1 point) Indicate whether plants A and B are C3 or C4 plants. Indicate the correct answers
with a tick () in the Answer Sheet.

Q2.16 (2 points) What is the net photosynthetic CO2 assimilation rate for Plant A and Plant B
measured at 200 l l-1 of CO2 concentration?

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PRACTICAL TEST 3

Q2.17 (2 points) Plot another graph by using CO2 assimilation rate from 20 to 100 l l-1 of CO2
concentration only (i.e., at low CO2 concentrations) for Plant B in Graph 2 provided in the
Answer Sheet. Use an X-axis scale from 0 to 100 l l-1.

Q2.18 (1 point) Based on Graph 2, what is the CO2 compensation point for plant B? Write the
value in the Answer Sheet.

Q2.19 (1 point) Compared to the data in Graph 2, would the CO2 compensation point increase,
decrease or remain unchanged if the measurements were carried out at 35 C and 21% O2?
Indicate the correct answer(s) with a tick () in the Answer Sheet.

Q2.20 (1 point). Compared to the data in Graph 2, would the CO2 compensation point increase,
decrease or remain unchanged if the measurements were carried out at 25 C and 2% O2?
Indicate the correct answer(s) with a tick () in the Answer Sheet.

END OF PAPER

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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

Country: _____________________

Student Code: ________________

23rd INTERNATIONAL BIOLOGY OLYMPIAD

8th 15th July, 2012
SINGAPORE

PRACTICAL TEST 4
ANIMAL ANATOMY & ECOLOGY
Total points: 100

Duration: 90 minutes

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IBO2012
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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

Dear Participants

In this test, you have been given the following two tasks:
Task I: Anatomy of molluscs. (20 points)
Task II: Rank-abundance plots, ABC curves and community structure. (80 points)

Use the Answer Sheet, which is provided separately, to answer all the questions.

The answers written in the Question Paper will NOT be evaluated.

Write your answers legibly in ink.

Please make sure that you have received all the materials and equipment listed for each task.
If any of these items are missing, please raise your hand immediately.

Stop answering and put down your pen IMMEDIATELY when the bell rings.

At the end of the test, place the Answer Sheet and Question paper in the envelope provided.
Our Assistants will collect the envelope from you.

Have fun and Good Luck!

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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

Materials and equipment:

For Task I: Anatomy of molluscs
Materials and equipment

Quantity

Unit

Mollusc 1 (in vial)

specimen

Mollusc 2 (in vial)

specimen

stereomicroscope

set

scissors

pair

forceps

pairs

plastic tray

piece

1000

mL

sheet

water (in beaker)

paper towels

For Task II: Rank-abundance plots, ABC curves and community structure
Materials

Quantity

Unit

Community 1

bag

Community 2

bag

Table A

sheet

Table B

sheet

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IBO2012
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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

Task I (20 points)

Anatomy of molluscs

Introduction
Members of the class Bivalvia are successful molluscs with a long evolutionary history. They
possess hinged left and right shell valves that enclose a headless animal within.
Vials labelled 1 and 2 contain two species of marine bivalves that are common in tropical Asia but
live in different habitats. The specimens were partly boiled and preserved in 70% ethanol.
Follow the instructions below to open the animals up for detailed examination under the
stereomicroscope.

Locate the anterior (if present) and posterior adductor muscles that join the left and right
valves of the animal.

Use the pair of scissors to cut the adductor muscles so that the valves can be separated to
expose the internal parts of the animal.

Observe the specimens under water in the trays provided.

Answer the following questions in the Answer Sheet:

Q1.1

(3 points 2 = 6 points) In which habitat (a d) would you expect to find species 1 and 2
respectively?
a.

attached to rocks or other hard surfaces

b.

boring into coral

c.

buried in sand or mud

d.

lying unattached on a sandy substratum

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Q1.2

PRACTICAL TEST 4

(2 points 2 = 4 points) How many pairs of ctenidia (gills) are there in species 1 and 2
respectively? Answer using numerals.

Q1.3

(2 points 2 = 4 points) How many pairs of labial palps are there in species 1 and 2
respectively? Answer using numerals.

Q1.4

(2 points 2 = 4 points) Locate the anus near the posterior end of the animal in each
species. The anus empties its contents into the path of the exhalant water flow. Starting
with the anus, trace the path of the intestine forwards towards the stomach. Indicate the
position of the intestine in relation to the heart (a e) in the two species respectively.
a.

intestine passes dorsally over the heart

b.

intestine passes under (ventral to) the heart

c.

intestine passes through the heart

d.

intestine passes to the right of the heart

e.

intestine passes to the left of the heart

Q1.5

(0.4 points 5 = 2 points) The following is a list (a-e) of anatomical features in

molluscs. Indicate with a tick () if the feature may be present in bivalves and with a cross
() if it is always absent.
a.

crystalline style

b.

eye

c.

foot

d.

penis

e.

radula

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PRACTICAL TEST 4

Task II (80 points)

Rank-abundance plots, ABC curves and community structure

Introduction
Changes in community structure may be visualized using a variety of graphs. A rank-abundance
plot or Whittaker plot is used by ecologists to display relative species abundance, a component of
biodiversity. In this type of graph, the rank of each species is plotted along the X axis. The most
abundant species is ranked 1, the second most abundant species is ranked 2, and so forth. The
abundance of each species is plotted on the log scale of the Y axis. The shape of the curve can
provide an indication of dominance or evenness.

The Abundance-Biomass Comparison (ABC) method was proposed by Warwick (1986) as a

technique for monitoring disturbance on benthic invertebrate communities. ABC curves have a
theoretical background in classical theory of r- and K-selection. The relative positions of the
abundance curve and biomass curve serves to indicate the level of disturbance in the community
(see graph below).

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Q2.1

PRACTICAL TEST 4

(16 points 2 = 32 points) In an environmental impact assessment (EIA) study on the

impact of salmon cage farming on benthic communities, samples were collected from the
various stations along two transects, A1 to A3, and B1 to B7 (see figure below).

, location of floating salmon cages;

, indicates direction of current flow along the coast.

You are a summer intern at the marine laboratory in which this EIA study is conducted. Your
responsibilities include the processing of benthic samples. You are given two bags
containing Community 1 and 2 and your job is to process the samples and collate
information similar to that carried out by a senior research assistant for Community 3 and 4
(see Table A, page 11 for Summary of the information). Each community may contain any of
the 17 species (A Q) listed in Table B (page 12); the respective mean fresh biomass per
individual of each species is also provided in Table B.

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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

The abundance of each species in Community 1 and 2 is indicated with different-sized chips
(see photograph below); e.g., there are 61 individuals of species A shown here:

1 individual

10 individuals

50 individuals

Determine the abundance of each species in Community 1 and record your data in
Table 1 in the Answer Sheet.

Fill in the rest of the required information (to 2 decimal places) in the table. Please note
that lg in the table and figure represents log10 and in the calculator, this is represented
by the log button.

Q2.2

Repeat the entire procedure for Community 2.

(3 points 4 = 12 points) Using your data in Tables 1 and 2, as well as the data provided
in Tables 3 and 4, plot the rank-abundance curves for Community 1 to 4 on Graphs 1 to 4
provided in the Answer Sheet.
Answer the following questions in the Answer Sheet. Indicate correct answer(s) with a tick
() and incorrect answer(s) with a cross ().
Q2.2.1 (1 point 5 = 5 points) Low evenness is:
a.

indicated by a steep slope in the rank-abundance curve.

b.

shown in Community 1.

c.

shown in Community 2.

d.

shown in Community 3.

e.

shown in Community 4.

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PRACTICAL TEST 4

Q2.2.2 (2.5 points 4 = 10 points) Indicate the correct ABC curves (A H) that
correspond to Community 1 to 4.

abundance; biomass

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ANIMAL ANATOMY & ECOLOGY

PRACTICAL TEST 4

Q2.2.3 (1.5 points 4 = 6 points) Rank Community 1 to 4 in decreasing levels of

disturbance.

Q2.2.4 (1 point 10 = 10 points) A careless summer intern, mixed up the labels for the
sampling stations (A1 A3; B1 B7) when the samples were transferred from
leaking containers to new bottles. From which stations could the benthic samples
containing Community 1 to 4 likely to be collected?

Q2.2.5 (2.5 points) Which of the 17 species (A Q) is likely to be a bivalve?

Q2.2.6 (2.5 points) Which of the17 species (A Q) has the potential to be a bioindicator of
organic enrichment?

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PRACTICAL TEST 4
Table A

Community 3

Community 4

Species

Abundance

Rank

Log10 (lg)
Abundance

Cumulative
%
Abundance

Cumulative
%
Biomass

Species

Abundance

Rank

Log10 (lg)
Abundance

Cumulative
%
Abundance

Cumulative
%
Biomass

200

2.30

14.31

28.30

320

2.51

60.49

7.83

180

2.26

27.18

41.03

78

1.89

75.24

38.37

175

2.24

39.70

50.76

50

1.70

84.69

60.39

150

2.18

50.43

58.34

32

1.51

90.74

71.35

120

2.08

59.01

68.65

25

1.40

95.46

85.42

112

2.05

67.02

73.74

10

1.00

97.35

91.53

98

1.99

74.03

81.17

0.70

98.30

93.98

80

1.90

79.76

86.02

0.60

99.05

96.62

75

1.88

85.12

88.29

0.48

99.62

98.53

62

10

1.79

89.56

92.36

10

0.30

100.00

100.00

35

11

1.54

92.06

94.40

30

12

1.48

94.21

96.45

28

13

1.45

96.21

97.44

25

14

1.40

98.00

98.45

15

15

1.18

99.07

98.48

16

0.90

99.64

98.99

17

0.70

100.00

100.00

Total

1398

Total

529

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PRACTICAL TEST 4

Table B. Fresh biomass per individual for species A to Q.

Species

Mean fresh biomass (g)

0.70

0.90

1.40

2.80

1.30

1.15

0.80

1.35

1.25

0.05

4.00

1.10

1.50

1.00

0.60

1.70

1.20

END OF PAPER

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