New Senior Secondary Mastering Biology (Third Edition)

Practical Worksheets for SBA E3

Suggested answers to Practical Worksheets

Ch 1 Basic microbiology

Practical 1.1 Culture of yeast using aseptic techniques

Understanding procedure (p. 139)
1 To kill any microorganisms present on the work surface.

2 To kill any microorganisms present on the inoculating loop.

3 To create an upward current of air to prevent any microorganisms in the air from getting
into the tube.

4 To minimize the exposure of the agar plate to the air and reduce the chance of
contamination.

5 Digging the loop into the agar not only damages the agar but also reduces the growth of
yeasts as the yeast cells are deposited in a less aerobic environment.

6 To prevent water vapour from condensing on the lid and dripping down of water droplets
onto the agar surface during incubation.

Results (p. 139)

B Culture of yeast in nutrient broth

Appearance of nutrient broth

Before incubation After incubation

Turbidity
Clear Turbid
(turbid / clear)

Pellicle
Absent Present
(present / absent)

Sediment
Absent Present
(present / absent)

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C Culture of yeast on a nutrient agar plate

Appearance of yeast colony

Shape (e.g. circular, irregular) Circular

Colour Cream colour

Surface (e.g. smooth, rough) Smooth

Elevation (e.g. flat, convex) Flat

Margin (e.g. smooth, lobate) Smooth

Opacity (e.g. transparent, opaque) Opaque

Questions (p. 140)

1 The culture of yeast may be contaminated with microorganisms from handlers and the
environment. The colonies may have different colours due to the presence of more than
one species of microorganisms. / Yeast may not grow well due to competition with other
unwanted microorganisms.

2 The nutrient broth provides a liquid medium and all the nutrients needed for the growth of
baker’s yeast.

3 Each colony represents one living yeast cell from the original sample.

4 When the streak was first made on the agar, the number of yeast cells in it was still very
large. The yeast cells grew close to each other and merged into a line.

5 This indicates the nutrient agar plate had been contaminated.

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Practical 1.2 Design an investigation to study the growth of

microorganisms
Aim (p. 142)
To investigate the growth of yeast in a closed system.

Introduction (p. 143)

1 Principle
To estimate the growth of yeast, we can measure the population size of yeast in a liquid
culture at different time. The population size of yeast can be estimated by performing a
viable cell count. After preparing a series of dilutions of the yeast culture and incubating
the agar plates with a known volume of each diluted culture, the number of living yeast
cells present in the original culture at different time can be found.

a Identification of variables
i The period of time for the growth of yeast. Incubate the yeast culture for
different periods of time.

ii The number of living yeast cells. A viable cell count is performed to determine
the number of living yeast cells.

iii Temperature, pH value, supply of nutrients, oxygen availability, water

availability, etc.

b Control
No. The investigation aims to compare the numbers of yeast cells in the liquid culture
at different time.

c Assumptions
All living yeast cells in the liquid culture form colonies.
Each colony arises from one yeast cell only.

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Materials and apparatus (p. 144)

Bunsen burner 1
test tube rack 7
test tube with stopper 24
micropipette with sterile tips (1000 μL) 1
micropipette with sterile tips (200 μL) 1
spreader 4
nutrient agar plate 24
transparent plastic bag 24
disposable gloves 1 pair
laboratory coat 1
waterproof marker pen 1
sticky tape
liquid culture of baker’s yeast
nutrient broth
70% alcohol
vortexer 1
incubator 1
autoclave 1

Procedure (p. 144)

1 Clean the work surface with 70% alcohol.
2 Sterilize the nutrient broth, the agar plate and the apparatus in the autoclave.
3 Label four test tubes A to D.
4 Attach a micropipette tip to the micropipette firmly. Keep holding the micropipette. Use
another hand to shake the tube of liquid culture of baker’s yeast gently.
5 Remove the stopper with your little finger. Flame the mouth of the tube.
6 Transfer 1 cm3 of the original yeast culture into 9 cm3 of nutrient broth in test tube A using
the micropipette.
7 Mix the contents well. Transfer 1 cm3 of the diluted yeast culture from test tube A into
9 cm3 of nutrient broth in test tube B.
8 Mix the contents well. Transfer 1 cm3 of the diluted yeast culture from test tube B into
9 cm3 of nutrient broth in test tube C.
9 Mix the contents well. Transfer 1 cm3 of the diluted yeast culture from test tube C into
9 cm3 of nutrient broth in test tube D.
10 Transfer 0.1 cm3 of each of the diluted yeast culture in test tubes A to D to four labelled
nutrient agar plates.
11 Spread the diluted yeast culture over the surface of each agar plate evenly using a sterilized
spreader.
12 Cover the agar plates well and seal the plates with sticky tape.
13 Put the tube of original culture into an incubator set at 30 °C.
14 Incubate the plates upside down with the same incubator for 24 hours.

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15 Choose a plate with 30–300 colonies for counting. Count the number of colonies on the
agar plate.
16 Repeat steps 3 to 15 every 24 hours for five days.
17 Calculate the number of living yeast cells in the original culture using the formula below:
1
Number of living yeast cells (per cm3) = Number of colonies × × Dilution factor
0.1
18 Plot the log10 number of living yeast cells against time.

Results (p. 145)

Number of living yeast Log10 number of
Time (h) Number of colonies cells in the original culture living yeast cells
(per cm3) (per cm3)

24

48 (Results vary with Ss.)

72

96

120

Discussion (p. 145)

1 The growth curve of yeast is S-shaped. The curve shows four phases:
During the lag phase, there is only a small increase in the number of yeast cells. The cells
are adapting to the new environment and preparing for growth.
During the exponential phase, the cells divide at the maximum rate and the population
increases exponentially. This phase continues as long as the supply of nutrients is adequate
and other conditions are favourable.
During the stationary phase, the population becomes constant and reaches a dynamic
equilibrium. The rate of formation of new cells equals the death rate. The large population
consumes large amounts of nutrients and releases toxic waste into the nutrient broth. A
depletion of nutrients and an accumulation of toxic waste make the conditions less
favourable for growth. Therefore, the growth rate slows down.
During the death phase, the population declines. With a further decrease in the amounts of
nutrients and further accumulation of toxic waste, the environment becomes harsh for
growth. The rate at which cells die exceeds the rate at which they are produced.

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2 The lag phase in the growth curve of the yeast would be missing. This is because the yeast
does not need to adapt to the new environment. The yeast would reach its maximum
growth rate once it is transferred to the new medium.

3 The liquid culture may be contaminated with microorganisms in the environment during
the practical. / The distribution of yeast cells in the liquid culture may be uneven.

4 Follow the aseptic techniques strictly during the practical. / Mix the liquid culture well
before each transfer the culture.

Conclusion (p. 146)

The growth curve of yeast is S-shaped. The curve shows four phases: the lag phase, the
exponential phase, the stationary phase and the death phase.

Ch 2 Use of microorganisms

Practical 2.1 Production of alcoholic drinks and bread by

fermentation
Understanding procedure (p. 151)
1 To allow the carbon dioxide produced to escape from the boiling tubes.

2 To provide a warm environment for yeast to ferment the sugars.

Results (p. 152)

A Production of alcoholic drinks

Extract Smell of the mixture

Apple Like alcohol

Orange Like alcohol

Grape Like alcohol

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B Production of bread
Observation

Change in the volume

of the dough Increases
(increases / decreases)

Texture inside the

bread Spongy
(spongy / hard)

Discussion (p. 152)

1 The extracts of apples, oranges and grapes contain sugars. The yeast ferments the sugars
into ethanol.

2 The volume of the dough increases. This is because the sugars in the dough are fermented
by yeast. The carbon dioxide produced forms bubbles inside the dough and causes it to
rise.
The texture inside the bread is spongy. This is because the bubbles of carbon dioxide
trapped by the dough expand upon heating. This gives the bread a spongy texture.

Practical 2.2 Design an investigation of the optimal conditions for

fermentation by yeast
Aim (p. 154)
To investigate the effect of the types of sugars used / temperature / pH on the rate of
fermentation by yeast.

Introduction (p. 154)

1 Problem
How do / does the types of sugars used / temperature / pH affect the rate of fermentation by
yeast?

2 Principle
Yeast carries out alcoholic fermentation under anaerobic conditions. During this process,
glucose is broken down into ethanol and carbon dioxide. The volume of carbon dioxide
released in a certain period of time gives the rate of fermentation by yeast. By comparing
the rate of fermentation in different conditions, the optimal conditions for fermentation by
yeast can be found.

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a Identification of variables
i (Answer varies with the condition being investigated.)

ii (Answer varies with the condition being investigated.)

iii (Answer varies with the condition being investigated.)

b Control
No. The investigation aims to compare the rate of fermentation by yeast with
different types of sugars used / at different temperature / at different pH values
(depending on the condition being investigated).

c Assumptions
The distance travelled by the water droplet is only due to carbon dioxide produced
from fermentation by yeast. / In the investigation of the effect of temperature, the
expansion of carbon dioxide due to any increase in temperature is negligible.

Materials and apparatus (p. 156)

beaker (50 cm3) 5
pipette (1 cm3) 5
syringe (5 cm3) 5
glass rod 5
pipette filler 1
stand and clamp 5
stop-watch 1
water bath at different temperatures 5
(e.g. 0 °C, 15 °C, 30 °C, 45 °C and 60 °C)
different types of 2% sugar solution
(e.g. glucose, lactose, fructose, galactose
and maltose)
buffer solution at different pH values
(e.g. pH 2, 4, 6, 8 and 10)
liquid culture of baker’s yeast
distilled water

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Procedure (p. 156)

1 Set up the apparatus as shown on the right.
2 Depending on the condition being
investigated, add the solutions below into the
mixture in the syringe or put the set-ups in
the place below:
 Types of sugars used – use different types
of sugars (e.g. glucose, lactose, fructose,
galactose and maltose) to prepare the
sugar solutions
 Temperature – put the set-ups into water
baths at different temperatures (e.g. 0 °C,
15 °C, 30 °C, 45 °C and 60 °C)
 pH – use buffer solutions at different pH
values (e.g. pH 2, 4, 6, 8 and 10) instead
of distilled water
3 Allow the mixture to incubate for five minutes.
4 Record the initial position of the water
droplet.
5 Record the final position of the water droplet after a certain period of time (e.g.
15 minutes).
6 Re-adjust the water droplet and repeat the experiment in different conditions.
7 Calculate the rate of fermentation by yeast.

Results (p. 157)

Volume of gas released Rate of fermentation
Condition investigated
in 15 minutes (cm3) by yeast (cm3/min)

(Results depend on the condition being investigated.)

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Discussion (p. 157)

1 (Answer varies with Ss.)

2 (Answer depends on the design.)

3 (Answer depends on the design.)

Conclusion (p. 158)

(Answer varies with Ss.)

Practical 2.3 Production of yoghurt by fermentation

Understanding procedure (p. 161)
1 To kill any microorganisms present in the milk.

2 To prevent the lactic acid bacteria from being killed by the high temperature.

Results (p. 162)

Observation

Texture of the mixture Thickens

Smell of the mixture Sour

Discussion (p. 162)

The texture of the mixture thickens. This is because the lactose in the milk is fermented by
lactic acid bacteria. The lactic acid produced lowers the pH of the milk. The milk proteins
therefore coagulate and the milk thickens to become yoghurt. The smell of the mixture is sour
due to the lactic acid produced in the mixture.

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