Core practical 12 Teacher sheet

EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Core practical 12: Investigate the rate of growth of

microorganisms in liquid culture

Objectives
● To understand how microorganism growth rate in liquid culture can be measured
● To be able to culture microorganisms with due regard for safe practice

Safety Specification links

● The culture of yeast is a possible biological ● Practical techniques 1, 3, 8, 9, 12
hazard as there is potential for ● CPAC 1a, 2a, 3b, 4a, 4b, 5a
contamination. A single yeast culture flask
should be set up for the class.
● If samples are taken this should be over a
period of no more than 12 hours.
● Wear eye protection.
● Use aseptic techniques when transferring
the yeast to or from the culture flask.
● Disinfect the bench with 1% Virkon® or
equivalent. Leave the disinfectant on the
bench for about 10 minutes. This should be
done before and after working.
● Ensure that all equipment is returned to the
designated point for sterilisation or disposal.
Procedure Notes on procedure
You will use an optical method to study the ● It is important that students have practised
growth of yeast in a liquid culture. The more the required aseptic techniques before the
yeast cells in a culture, the more turbid the practical task. Teachers should be familiar
culture. This means that less light can pass with microbiology techniques. Ensure that
through the sample. A colorimeter can be used good practice (e.g. flaming of culture flask
to measure turbidity in samples taken at regular neck, use of sterile materials, stoppering of
intervals. The absorbance of light is measured. culture flask with non-absorbent cotton
This is sometimes also called optical density or wool, autoclaving of equipment and culture
OD. If using a light sensor and datalogger a light flask) is carried out.
is shone through the culture, and the light ● Plan the timing of this practical activity
coming through to the other side is measured carefully, especially if students will be using
using a light meter or sensor which is connected a colorimeter.
to a datalogger and recorded automatically over
time. ● A single culture flask should be set up for
the whole class and inoculated with yeast
Steps 4 to 6 provide the data required to draw during the task introduction. Incubate at
an indirect growth curve for the culture and room temperature (no greater than 25 °C).
calculate the exponential growth rate constant.
Steps 7 to 12 are required to calculate yeast cell ● Two different methods are described:
numbers (as cell densities) which can be used to measuring changes in turbidity using either
produce a direct growth curve showing cell a) a colorimeter or b) a light sensor plus
numbers. datalogger.
It is important to follow aseptic technique
throughout to avoid contamination of your If using a colorimeter
cultures, of yourself and of the surrounding ● The absorbance of samples should be
environment. The method below includes some measured initially and then at timed
precautions but they are not exhaustive, intervals, ideally every 20–30 minutes for
requirements will depend to some extent on the the first couple of hours and then
method used. approximately hourly, completing at least
1. Wash your hands with soap and disinfect six measurements during a period of 5–12

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 Core practical 12 Teacher sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

your bench area, leaving it to soak for 10 hours. A culture period of 9–10 hours will
minutes if using Virkon®, before wiping it probably be needed to reach the stationary
down. Light a Bunsen burner set to a safety phase, but sufficient growth should occur
flame on the disinfected working area. within the first 5 hours to enable the
2. Your teacher will carry out this step to exponential growth rate to be estimated. A
produce a single culture flask for the whole yeast culture could be set up in a morning
class. lesson and then sampled throughout the
day by different groups of students. Ensure
● Use a sterile clear-sided 500 cm3 conical adequate supervision of sampling if this
flask containing 250 cm3 of 0.5% takes place outside of normal lesson times.
glucose solution as the culture vessel.
This should be stoppered with cotton ● If the OD rises above an absorbance value
wool and should contain a sterile of 2, the accuracy will be lost. It may
magnetic stirring ‘flea’. therefore be necessary to dilute later
samples to bring them within the range.
● Using aseptic technique to prevent This dilution must then be accounted for in
contamination, add 1.25 g of yeast to calculating the absorbance: multiply the
inoculate your culture vessel. Swirl to reading by the dilution factor.
mix.
3. Place the flask on a magnetic stirrer and stir
continuously. Cover the cotton wool stopper If using a light sensor and datalogger
loosely with aluminium foil and incubate at ● With continual datalogging the timing issues
room temperature. can be avoided as there is no need to take
If using a colorimeter samples. Once the culture flask has been
set up there is no need to remove the cotton
4. Fill a cuvette with glucose culture medium wool stopper, so contamination and safety
and use this as a blank to set the risks are reduced. In this case the culture
absorbance of the colorimeter to zero. Keep can be left for up to 24 hours, provided that
the glucose solution (the blank) in a the culture flask is not opened during this
refrigerator between measurements. time and is transferred directly to the
5. As soon as the yeast suspension has been autoclave or pressure cooker.
mixed, use aseptic technique to measure a ● The exponential growth rate constant
3 cm3 sample into a clean measuring calculated from this method will be negative
cylinder. Transfer the sample immediately (because light levels decline). The sign of
into a cuvette (which should be just over the constant can be ignored.
half full) and measure the absorbance.
Record time and absorbance in a table.
Return the stoppered yeast culture to the Extension activity
stirrer. ● This allows students to calibrate results by
6. Repeat steps 4–6 at least five times over determining the cell density of the initial
the next 12 hours. Take samples at microorganism population. If using the
intervals of 20–30 minutes over the first 2 datalogger method there will be more time
hours. Do not remove any samples after 12 for students to carry out the extension
hours of culturing. activity.
If using a light sensor and datalogger ● Once cell density of the initial culture has
7. Place a cool light source to one side of the been determined, students can calculate
yeast culture. Place a light sensor at the the number of microorganisms that will
opposite side, very close to the culture result after a given time period during the
flask. logarithmic growth phase.
8. Place a carboard box, with a cut-out to let in Using the formula: Nt  N0  2kt
light from the light source, over the flask, Where:
stirrer and light sensor. This will protect the
sensor from ambient light. ● Nt is the number of organisms at time t
9. Connect the light sensor to a datalogger ● N0 is the number of organisms at time 0
and leave to record continuously for ● k is the exponential growth rate constant
between 10 and 24 hours. ● t is the time the colony has been growing.

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 Core practical 12 Teacher sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Extension – Relating the absorbance or light

readings to the density of yeast cells in
culture
10. The initial density of cells in the culture (for
1.25 g of yeast in 250 cm3 solution) can be
estimated using a haemocytometer and
manufacturer’s instructions.
11. Alternatively find the area of your
microscope’s field of view using a slide of
mm graph squares on acetate. Count the
squares visible using the ×4 objective. Now
calculate the area of the field of view for the
×40 objective which is 0.01 of the area of
the ×4 objective (i.e. the square of 4/40 as
this is area).
12. Stain the yeast suspension with a few drops
of 0.1% methylene blue. Using a 1 cm3
plastic pipette, place one drop of this yeast
suspension on to a microscope slide and
cover with a coverslip. View under the ×40
objective and count the yeast cells that you
can see within the one field of view.
13. Calculate the volume of one pipette drop by
measuring the volume of 10 drops. This
gives the volume under the area of the
coverslip. The volume under one field of
view of the ×40 objective can then be
calculated (divide the area of the ×40 field
of view by the area of the coverslip then
multiply this figure by the volume of one
drop).
14. Work out the initial yeast population cell
density (cells/mm3). This is average cell
count in one field of view divided by the
volume of that field of view.
15. If many cells overlap when viewed under
×40 objective, then counts will not be
accurate and dilution of the yeast
suspension may be required.
16. Wash your hands and disinfect surfaces
before leaving the laboratory.

Answers to questions
1. Calibrate OD readings using either dilution plating or direct counting (using a haemocytometer).
Produce a calibration curve for a range of OD readings and use the graph to estimate the
number of microorganisms present.
2. The method is valid because most of the change in turbidity will be a result of microorganism
cells in suspension. However, the method does not distinguish between live cells and other
particles in suspension such as dead cells, spores or cell fragments, which reduces the validity.
3. The magnetic stirrer keeps cells in suspension and ensures that nutrients are evenly
distributed. It also oxygenates the culture to allow aerobic respiration.

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 Core practical 12 Teacher sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Sample data

Time/hours OD culture 1 OD culture 2 OD culture 3

0.00 0.06 0.04 0.05
0.75 0.05 0.07 0.07
2.25 0.19 0.28 0.25
3.75 0.66 0.87 0.85
5.25 1.27 1.10 1.30
6.75 1.47 1.16 1.32
8.25 1.53 1.18 1.40
9.75 1.65 1.23 1.55
11.25 1.78 1.30 1.72
15.00 1.97 1.29 1.90
Table A Changes in optical density against time in three cultures of yeast Saccharomyces sp.
incubated at 25 °C.

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 Core practical 12 Student sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Core practical 12: Investigate the rate of growth of

microorganisms in liquid culture

Objectives
● To understand how microorganism growth rate in liquid culture can be measured
● To be able to culture microorganisms with due regard for safe practice

Safety All the maths you need

● The culture of yeast is a possible ● Recognise and make use of appropriate
biological hazard as there is potential for units in calculations.
contamination. A single yeast culture ● Recognise and use expressions in
flask will be set up for the class. decimal and standard form.
● If samples are removed this should be ● Estimate results.
over a period of no more than 12 hours.
● Use calculators to find and use power,
● Wear eye protection. exponential and logarithmic functions.
● Use aseptic techniques when ● Use an appropriate number of
transferring the yeast to or from the significant figures.
culture flask.
● Substitute numerical values into
● Disinfect the bench with 1% Virkon® or algebraic equations using appropriate
equivalent. Leave the disinfectant on the units for physical quantities.
bench for about 10 minutes. This should
be done before and after working. ● Use logarithms in relation to quantities
that range over several orders of
● Ensure that all equipment is returned to magnitude.
the designated point for sterilisation or
disposal. ● Translate information between
graphical, numerical and algebraic
forms.
● Plot two variables from experimental or
other data.
● Calculate rate of change from a graph
showing a linear relationship.
● Draw and use the slope of a tangent to
a curve as a measure of rate of change.
Equipment
● bench disinfectant, paper towels or cloth for the yeast culture (one for whole class):
● Bunsen burner ● dried baker’s yeast (1.25 g)
either ● 250 cm3 of culture medium (0.5%
● colorimeter set to absorbance (with filter glucose solution) in a 500 cm3 sterile
of around 600 nm wavelength) conical flask
● colorimeter cuvettes ● small amount of glucose culture medium
to blank the colorimeter
● 5 cm3 measuring cylinders or 2000 µl
automatic pipette ● sterilised magnetic stirrer
or ● non-absorbent cotton wool and
● light source, light sensor and datalogger aluminium foil
for counting cell densities:
● microscope, with slide and coverslip
● dropper pipette
● mm2 graph paper photocopied on to
acetate

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 Core practical 12 Student sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Diagram

fig A Turbidity measurements can be used to measure a population of microorganisms indirectly.

With a colorimeter, the higher the absorbance of light, the more yeast cells are present. Using a
light sensor and datalogger, the higher the transmitted light reading, the fewer yeast cells are
present.
Procedure
You will use an optical method to study the growth of microorganisms in a liquid culture. The more
yeast cells in a culture, the more turbid the culture. This means that less light can pass through the
sample. A colorimeter can be used to measure turbidity in samples taken at regular intervals. The
absorbance of light is measured. This is sometimes also called optical density or OD. If using a light
sensor and datalogger, a light is shone through the culture, and the light coming through to the
other side is measured using a light meter or sensor which is connected to a datalogger which
records automatically over time.
Steps 4 to 6 provide the data required to draw an indirect growth curve for the culture and calculate
the exponential growth rate constant. Steps 7 to 12 are required to calculate yeast cell numbers (as
cell densities) which can be used to produce a direct growth curve showing cell numbers.
It is important to follow aseptic technique throughout to avoid contamination of your cultures, of
yourself and of the surrounding environment. The method below includes some precautions but
they are not exhaustive; requirements will depend to some extent on the method used.
1. Wash your hands with soap and disinfect your bench area, leaving it to soak for 10 minutes if
using Virkon®, before wiping it down. Light a Bunsen burner set to a safety flame on the
disinfected working area.
2. Your teacher will carry out this step to produce a single culture flask for the whole class.
● Use a sterile clear sided 500 cm3 conical flask containing 250 cm3 of 0.5% glucose solution
as the culture vessel. This should be stoppered with cotton wool and should contain a sterile
magnetic stirring ‘flea’.
● Using aseptic technique to prevent contamination, add 1.25 g of yeast to inoculate your
culture vessel. Swirl to mix.
3. Place the flask on a magnetic stirrer and stir continuously. Cover the cotton wool stopper
loosely with aluminium foil and incubate at room temperature.
If using a colorimeter
4. Fill a cuvette with glucose culture medium and use this as a blank to set the absorbance of the
colorimeter to zero. Keep the glucose solution (the blank) in a refrigerator between
measurements.
5. As soon as the yeast suspension has been mixed, use aseptic technique to measure a 3 cm3
sample into a clean measuring cylinder. Transfer the sample immediately into a cuvette (which
should be just over half full) and measure the absorbance. Record time and absorbance in a
table. Return the stoppered yeast culture to the stirrer.
6. Repeat steps 4–6 at least five times over the next 12 hours. Take samples at intervals of 20–30
minutes over the first 2 hours. Do not remove any samples after 12 hours of culturing.
If using a light sensor and datalogger
7. Place a cool light source to one side of the yeast culture. Place a light sensor at the opposite

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 Core practical 12 Student sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

side, very close to the culture flask.

8. Place a carboard box, with a cut-out to let in light from the light source, over the flask, stirrer
and light sensor. This will protect the sensor from ambient light.
9. Connect the light sensor to a datalogger and leave to record continuously for 10 to 24 hours.
Extension - Relating the absorbance or light readings to the density of yeast cells in culture
10. The initial density of cells in the culture (for 1.25 g of yeast in 250 cm3 solution) can be
estimated using a haemocytometer and manufacturer’s instructions.
11. Alternatively find the area of your microscope’s field of view using a slide of mm graph squares
on acetate. Count the squares visible using the ×4 objective. Now calculate the area of the
field of view for the ×40 objective which is 0.01 of the area of the ×4 objective (i.e. the square
of 4/40 as this is area).
12. Stain the yeast suspension with a few drops of 0.1% methylene blue. Using a 1 cm3 plastic
pipette, place one drop of this yeast suspension on to a microscope slide and cover with a
coverslip. View under the ×40 objective and count the yeast cells that you can see within the
one field of view.
13. Calculate the volume of one pipette drop by measuring the volume of 10 drops. This gives the
volume under the area of the coverslip. The volume under one field of view of the ×40
objective can then be calculated (divide the area of the ×40 field of view by the area of the
coverslip then multiply this figure by the volume of one drop).
14. Work out the initial yeast population cell density (cells/mm3). This is average cell count in one
field of view divided by the volume of that field of view.
15. If many cells overlap when viewed under ×40 objective, then counts will not be accurate and
dilution of the yeast suspension may be required.
16. Wash your hands and disinfect surfaces before leaving the laboratory.
Analysis of results
1. Plot a graph of absorbance or light reading against time.
2. Identify the lag and exponential (log) phases of the growth curve on your graph.
3. From the exponential (log) phase of the growth curve, estimate the time taken in hours for the
population size to double.
4. Calculate the exponential growth rate constant using the formula:
log10 OD1  log10 OD0
k
log10 2  t
Where:
● OD0 is the absorbance or light reading at the start of the log phase
● OD1 is the absorbance or light reading after time t (use a reading towards the end
of log phase)
● t is the time the culture has been growing.
5. If you have time, re-plot the absorbance–time graph using a semi-logarithmic scale.
This can be done using appropriate graph paper, or more easily by entering the
results into a spreadsheet and using the graphing functions. In popular drawing
packages, this may be achieved by drawing the graph then clicking on the axis and
selecting ‘logarithmic scale’. Examine the two graphs and compare them with the
examples in Student Book Section 6.1.3, fig A.
Learning tip
● When we talk about the growth of microbe populations we are referring to the increase in
the number of cells. The growth rate of microorganisms can be expressed in terms of the
mean growth rate constant (k). This is equivalent to the number of generations per unit
time. Growth can also be described using the time taken for one generation. As bacterial
populations grow by binary fission, this is the same as the time taken for the population (or
optical density) to double.

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 Core practical 12 Student sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Questions
1. The simple optical method for examining growth gives no measure of the actual numbers of
cells. How can this method be improved to provide such information?
2. Comment on the validity of using turbidity measurements to follow the growth of populations of
microorganisms.
3. Why was the magnetic stirrer used?

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 Core practical 12 Technician sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

Core practical 12: Investigate the rate of growth of

microorganisms in liquid culture

Objectives
● To understand how microorganism growth rate in liquid culture can be measured
● To be able to culture microorganisms with due regard for safe practice

Safety
● Wear eye protection.
● The microorganisms are a potential biological hazard. Use aseptic techniques when working
with the yeast cultures.
● Disinfect the bench with 1% Virkon® or equivalent. Leave the disinfectant on the bench for
about 10 minutes. This should be done before and after working.
● Follow current best practice for handling microorganisms and sterilising equipment before and
after use. An autoclave or pressure cooker will be required for effective sterilisation of
equipment and guidelines for safe use should be carefully followed.

Equipment per student/group Notes on equipment

bench disinfectant 1% Virkon® or equivalent to be left for about 10
minutes on the bench
paper towels or cloth to dry off the bench
Bunsen burner one per group
colorimeter set to absorbance with a filter of Set the colorimeter to approximately 600 nm
wavelength of around 600 nm wavelength or use an orange/red filter.
colorimeter cuvettes Small volume cuvettes (normally 4 cm3). Sterilise
cuvettes in an autoclave after use.
light sensor or light meter This is an alternative to the colorimeter. The
datalogger datalogger method has both practical and safety
advantages and should be used if possible.
Ensure that the light source will not produce
cool light source heat, is within its latest PAT test range, and is
positioned so that it is not likely to fall over Set to
continuous recording
cardboard box The box will shade the sensor from ambient
light. Cut out an area to allow light from the light
source to enter. Place the box over the flask,
stirrer and light sensor.
dried baker’s yeast (not fast acting) 1.25 g for yeast culture plus 1.25 g to make up
an identical suspension for counting initial yeast
cells density.

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 Core practical 12 Technician sheet
EDEXCEL Biology B Teacher Resource Pack 2 Investigate the rate of growth of microorganisms in liquid
culture

250 cm3 of 0.5% glucose solution in a sterile 500 Use only glucose solution as the medium.
cm3 clear conical flask A single culture flask will be set up as a culture
vessel for the whole class to measure.
The flask should be stoppered with non-
absorbent cotton wool covered with aluminium
foil.
Ensure that the flask used can fit into the
sterilising equipment available at the school.
Flasks containing incubated cultures must not
be opened before they have been fully sterilised
by steam at 121 °C for 15 minutes in an
autoclave/pressure cooker
small bottle of 0.5% glucose solution for use as store in the fridge between sampling events
a blank
magnetic stirrer The flea should be sterilised in 70% ethanol for
5 minutes before and after use. Remove from
the flask after use using another magnet and re-
sterilise. Do not autoclave as this will
demagnetise it.
non-absorbent cotton wool to stopper the culture flask
aluminium foil to cover the stopper
5 cm3 measuring cylinder or 2000 µl automatic Two per group. Measuring cylinders must be
pipette sterilised in an autoclave after use.
Provide a disposal container for pipette tips.
microscope with ×4 and ×40 objectives
mm2 graph paper photocopied on to acetate a 2 cm strip of the acetate such be fixed to the
attached to a microscope slide slide using sticky tape
slides and coverslips
dropper pipette
0.1% methylene blue stain
Notes

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