POPULATION GROWTH OF BREAD MOULD

Modified by John Osborne, from Janet Lanza, Biology Department, University of Arkansas at Little Rock

ABSTRACT
In this laboratory exercise you will design and conduct an experiment on the growth rate of
bread mould in different environmental conditions.

OBJECTIVES
After completing this exercise, you will be able to
define r, K, limiting factor, exponential growth
calculate r
correctly design and conduct an experiment
correctly collect and display data
interpret the data correctly
suggest additional studies

INTRODUCTION
More offspring are produced than can survive. This statement is an important observation of
all plants, animals, and microbes. When resources are unlimited, population growth is
exponential; in other words, population size is multiplied by a factor in a certain time interval.
Bacteria are often used to show this type of growth (Fig. 1).

Fig. 1. This graph shows the increase over time in the

number of bacterial cells in a laboratory culture. Each
cell divides every 20 minutes and so the population
doubles every 20 minutes.

Physical conditions can affect population growth. For example, individuals of a certain species of
white butterfly grow and develop more slowly at 20 C than at 28 C. This slower development
results in a slower growth rate of the population because the generation time is longer. Other
factors that can affect population growth include pH and sunlight.

Availability of resources can also slow population growth, either because of increased mortality
or because of decreased reproduction. Fig. 2 shows the number (in thousands) of male fur seals
on an island off the coast of Alaska from 1910 to 1950. The points are the actual number of
seals. This population was depressed by hunting before 1925. When hunting controls were
introduced in 1925, the population grew but then growth levelled off after about 1935. Most
likely, some resource in the environment limited further growth. Resources that can limit
population growth in this fashion include food, water (especially for plants), and nesting sites.
 Fig. 2. The population size of fur seals
(as measured by the number of males)
increased after hunting was banned in
1925. However, population growth
levelled off about 1935.

Finally, interactions with other species (e.g., competitors, predators, parasites, pollinators, and
dispersers) can also affect population growth rate.

The upper limit to population size, generally set by effects from competition, parasitism, and
food and space limitations, is called the carrying capacity (K). In Fig. 2, K for the seal population
is around 10,000 male seals.

Mathematical Description Of Population Growth

Population growth can be described mathematically rather simply. Before you think about the
equation, though, consider that a population changes size only through birth and death and
that the new population size is the previous population size with the births added and the
deaths subtracted. (In this mathematical model we are not taking into account individuals who
might emigrate away from, or immigrate into a population.)

Because it can be important to compare among populations and among times, birth and death
rates are expressed on a per capita basis i.e., the number of births or deaths per individual in
the population. An example might be that there are 300 births in a population of 2500 in a year
in this case the birth rate is 300/2500 or 0.12 per year.

But it can be difficult to measure actual birth and death rates and so population biologists often
just measure the population size and express population growth as a change in population size
in a given time interval.

Thus, the first equation to consider simply shows that the change in the number of individuals in
a population in a given time interval is the population growth rate (r, which is the birth rate
minus the death rate) multiplied by the number of individuals in the population at the beginning
of the time interval. In mathematical terms, the equation looks like this:

N/t = rN
N is the number of individuals in the population, N is the change in the number of individuals,
t is the change in time (the time interval), and r is the population growth rate (= birth rate
death rate).

This equation can be used to describe population growth like the bacterial growth shown in
Figure 1. It is called exponential growth. Notice that the slope of the curve keeps increasing
more and more individuals are added to the population each generation.
This equation can be modified slightly to account for the slowing of population growth as the
population size increases. The best way to explain this modification in words is to say that the
rate of population growth is multiplied by a factor (1- N/K) that measures how close the
population is to K (carrying capacity). In mathematical terms, the equation looks like this:

N/t = rN(1 N/K)

Note that if the population size (N) is very small, the value of the expression in parentheses
(brackets) (1 N/K) is close to 1 and the value of the whole equation is the rapid increase in
population growth that you saw above. However, as N approaches K, the value of N/K
approaches 1, and the value of the parenthetical expression approaches 0, causing the value of
the whole equation to also approach 0. At K, N/t = 0, meaning that the change in population
size is 0 and the population size is stable.

In this exercise, you will design an experiment to compare the population growth rate of bread
mould in different environmental conditions. Although you wont count mould individuals, you
will be able to use colony size as a measure of population size. This lesson will help you
understand the nature of population growth, the limits to population growth, and the effect of
the environment on the rate of population growth.

BACKGROUND RESEARCH INFORMATION LINKS

All biology and environmental science books have sections that discuss exponential growth and
the concept of carrying capacity. General biology textbooks also may have sections on fungi.
Genetics textbooks may have sections on Neurospora because it was an important tool in early
genetics studies and because its genome has now been sequenced.

A number of different moulds grow on bread,

including Rhizopus, Aspergillus, Neurospora,
and Penicillium. It is not so important in this
investigation to identify the mould species, but it might
be something interesting you want to do.

Fungi are interesting organisms, with structures and life

cycles different from other organisms. Again, no-one is
requiring you to have a deep understanding of fungal
biology (mycology) but you might be interested enough
to want to go further.

A variety of websites will provide good background information. Good key words to use in
searching this topic include bread mould, exponential growth, and population growth.

Some websites that provide relevant information include:

http://www.ecofuture.org/pop/facts/exponential70.html
http://pmo-sun.uoregon.edu/~dmason/stat/expgrow.html
http://www.fi.edu/tfi/units/life/forums/living/bread.html
http://www-genome.wi.mit.edu/annotation/fungi/neurospora/neurospora.html
http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Contents.html
MATERIALS
A variety of materials will be available for your use in this exercise. Every group will need the
items in A: Essential Items but different groups will use different items from B: Optional
Items.

A: Essential Items
bread
plastic sandwich bags (one for each piece of bread)
bread mould (provided)
cotton swabs or toothpicks (up to 1 per slice of bread)
paper towels
alcohol (isopropyl alcohol or 70 % ethanol) or 10 % bleach (enough to wipe down the
outsides of the plastic bags and your desks at the end of the lab)
nose/mouth masks for anyone allergic to moulds
masking tape (to seal bags if they are not self-sealing)
permanent marker (for labelling)

B: Optional Items
10 % NaCl or other salts (or other concentrations)
water, pH 5 (or other)
water, pH 9 (or other)
sugar (dry or in solution)
salt (dry or in solution)
drierite (anhydrous calcium sulphate) (to absorb water vapor) do not touch this use
a spatula or spoon
KOH (to absorb CO2) do not touch use a spatula or spoon
dry ice (to add CO 2) do not touch use tongs
laboratory gloves

PROCEDURE
Your task is to measure and compare the population growth of bread mould in two different
environmental conditions (two treatments). You will inoculate slices of bread with bread mould
cultures, place each slice of bread in a bag, and measure growth of the bread mould colonies
each day for a week.

You need to have two groups of bread slices each group under different experimental
conditions. Each person in the group will have at least one slice of bread from each treatment.
Variables that you can potentially manipulate include bread type, temperature, light, pH of the
bread surface, water content of the bread, humidity inside the bag, added sugar, added salt, and
gas (CO2) content. Maybe you have other suggestions?

LAB SAFETY
1. Are you allergic to moulds? You may have a partner inoculate your bread for you. Make
sure to seal the bag totally and wipe the outside of the bag with alcohol or 10 % bleach
when you are finished.
2. Keep your hands away from your face while working in the lab and wash your hands at
the end of each session.
3. Do not open the plastic bag containing the bread after the bread mould has started to
grow.
4. Do not touch drieite, dry ice or KOH pellets (if available).
5. Use plastic gloves if they are provided.
METHOD
The steps below represent general methods that you can use. If you think you have better
methods, discuss them with John before trying them out. You will need to decide what
environmental factor to vary and brainstorm a way to vary the factor. You will also need to
consider how to standardize your methods.

1. Work in groups of 2-4.

2. Brainstorm and decide what environmental factor you wish to vary and what methods you
can use for varying the factor.
3. Complete the Planning format and share it with John, for approval.
4. Collect your supplies at least two pieces of bread and two plastic bags per person,
masking tape to seal each bag, several paper towels, several cotton swabs or toothpicks,
and supplies for your manipulation. You should plan to have a minimum of four pieces of
bread per treatment. A larger sample size can give you more robust results but be guided
by the amounts of materials available.
5. Make sure to minimize the contamination of your bread by following the following
suggestions.
i. Get everything ready before you start to inoculate the bread.
ii. Keep the bags with mould cultures closed as much as possible.
iii. When you inoculate your bread (step 7), put it in its bag quickly so that you can limit
the number of spores that fall on the bread.
iv. If available, wear gloves when handling the bread.
6. Find a way to moisten the bread. One simple way is to wet a paper towel and place it on the
lab bench. Then place one piece of bread on the paper towel for a fixed length of time.
Next, remove the bread from the paper towel. Can you think of a better way to standardize
this step?
7. To inoculate your slice of bread, place a cotton swab or toothpick in a bread mould culture
and dab it onto a spot on the pre-moistened side of the bread. Repeat this step in five
different places on each slice of bread. How can you standardize this step? As quickly as you
can, place each piece of bread in its own plastic bag.
8. Close or tape the plastic bag closed (all open edges) and wipe the outside of the plastic bag
with alcohol or 10 % bleach. Also wipe down the countertop with alcohol or 10 % bleach.
Note that you never need to open the bag.

Tips For Designing Your Experiment

1. Make sure you vary (manipulate) only one factor.
2. Ask one simple, small question, not a large complex one.
3. Make sure everyone in the group knows what you are measuring/counting so that everyone
will make the same measurement.

DATA/OBSERVATIONS - RESULTS TABLE

Measure the size of each mould colony daily. You will need to decide how to measure colony
size. Note that colonies will not always be perfect circles. Keep track of each colony individually.
Make sure that everyone in the group measures colony size in the same way. Enter the result
into a table.
 Colonies of mould growing on bread. Make sure that
you find a way to make accurate quantitative
measurements of the fungal colonies.

ANALYSIS
Once you have all your data you will need to reduce your data. This process summarizes your
data so that you can see trends that will allow you to draw conclusions. The steps below will
help you with this process.

Data reduction
1. After one week, calculate the mean (average) diameter of mould colonies for each slice of
bread for each day. This will give you one number for each day for each of your pieces of bread.
2. Calculate mean (average) colony size for each day, using all the pieces of bread in a single
treatment.

Graphical analysis line graph

1. Make a line graph of colony size vs. time for each treatment. Make sure to think about which
measurements belong on the y and x axes.
2. Using the overall averages, calculate r for each treatment for each day. Instead of actual
numbers of individuals, you will use your measure of colony size as a measure of population
size. Calculate r as the slope of the curve (colony sizeday n+1 colony sizeday n). Make a table
showing slopes for all your graphs.
3. Did the slope of the lines for the two treatments differ? If so, is the difference what you
expected?
4. Did the slope of the lines change over time? If so, how? What happened to population growth
over time?
5. Answering questions 3 and 4 requires some thought. Ideally you would use a statistical test to
compare colony size on a given day or slopes of the lines but this is a rather complicated
procedure and you will probably not be able to conduct such an analysis. Nevertheless, you
need to examine these graphs carefully.

Graphical analysis bar graph

1. You should also make a bar graph of your results. To do this, look at the data for the faster-
growing treatment (if you have one). Then find the day that had the largest colonies without the
colonies touching each other. Use the data from this day to draw a bar graph showing mean
(average) colony size of the two treatments. Before you graph your data, identify your
manipulated and measured variables and think about which one goes on the y axis and which
one goes on the x axis.
2. These data are more amenable to statistical analysis than the data that you examined in
drawing the line graph. You could use a Students' t-test to analyze these data. A relatively easy
way to conduct this analysis is to use a web-based statistical program. One that I have
successfully used, and that has text to help you understand various statistical tests, can be found
at: http://faculty.vassar.edu/lowry/VassarStats.html.
Statistical analysis
Variability abounds in systems studied by many scientists. This variability necessitates the use of
replication in experiments and the use of statistics in data analysis. (If there were no variability,
we could conduct only one trial and know an answer.)

Statistics can be intimidating but the basic idea is actually easy to understand. The availability of
computers and statistical packages makes it very easy to conduct statistical tests (but care must
be exercised to know which test to use). But most important is knowing how to interpret a result
from a statistical test.

The easiest way to think about statistics is to ask yourself the following question, What is the
likelihood that differences between two groups are solely due to random variation in the
system? If differences are small, random variation may well account for them. If differences are
big (and you have controlled your conditions correctly), random variations probably cant
account for the differences the cause is likely to be your experimental variable!

Two answers are possible to your question:

1. The likelihood that the differences between the two groups is solely due to random variation
is high. This statement means that you will conclude that your experimental variable had little
effect. Thus, differences between the two groups are not significant.
2. The likelihood that the differences between the two groups is due to random variation is
low. This statement means that you will conclude that your experimental variable caused the
differences between the two groups. Thus, differences between the two groups are significant.

QUESTIONS
Be prepared to answer the following questions.
1. What was your experimental question?
2. How did you manipulate your treatments?
3. What kinds of standardizations did you employ?
4. Did you see evidence of exponential growth? of limitations of resources? Explain.
5. Summarize the results of your experiment. Did the different environmental conditions affect
the population growth of bread mould?
6. What three additional studies would be interesting to conduct?