Inhibition of the growth and reproduction of fungi

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HKTA The Yuen Yuen Institute No.2 Secondary School
Inhibition of the growth and reproduction of
fungi

Member Choi Wai Kwan 6S (2) Lam Oi Chu 6S
Biology Project
Dec. 2002
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Content Introduction Procedure
Result Errors and
Improvements Another design of experiment for the
same aim Background Information
Other alternatives of the experiment Further
Information
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Introduction Some of the plants can secrete a
special substance to kill the bacteria this
function can prevent the plants being invading by
bacteria in the soil or in the air. When the
plant is dead, bacteria to form the inorganic
substance will decompose this kind of special
substance. In the following experiment, we want
to investigate if the smell and the extract of
specific food such as wasabi, ginger, garlic, and
pepper can inhibit the growth and reproduction of
fungi.
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Procedure
1.First a piece of bread was prepared to culture
the mould for two weeks by adding water onto the
bread. And it was put in to a plastic bag to
prevent another kinds of spores of mould grow on
the bread. 2.After the mould was cultured on the
bread, it was cut into 9 pieces with equal area
(2 cm x 2cm)
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3.Use another bread (5 cm 5 cm) and the middle
area of the bread was cut to form an empty square
(2 cm x 2 cm). This step was repeated to prepare
9 pieces of such bread.
4. By using a forceps, each piece of bread (2 cm
x 2cm) on which the mould is culture was placed
into the empty space of the bread prepared in
step 3. And it was placed in the petri dish
covering lid.
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Precaution This step should be done immediately
to prevent another kinds of mould adding to the
bread.
5.To prepare the extract of different food, the
procedure was done as follows Extract of
wasabi It was prepared by adding 10 drops of
distilled water
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Extract of ginger and garlic Use a half of
garlic and ginger, which were peered. They were
cut into small pieces and grinded to obtain the
extract with the aid of pestle and mortar. And
they were added with 10 drops of distilled water.
Extract of ginger and garlic Use a half of
garlic and ginger, which were peered. They were
cut into small pieces and grinded to obtain the
extract with the aid of pestle and mortar. And
they were added with 10 drops of distilled water.
Extract of pepper It was prepared to grind the
pepper and be added with 50 drops of distilled
water.
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6.The extract of each kind of food was added onto
the bread prepared by using a dropper.
7.To prepare the smell of different food, the
procedure is done as follows Wasabi, several
pieces of garlic, ginger, 10 grains of pepper
were placed in the petri dish near the bread
respectively.
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8.The result was observed and recorded by taking
photos for every 3 days.
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mould
pepper
wasabi
garlic
ginger
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Results The Effect of solution of different food
extracts and its smells on the growth of bread
moulds
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Explanation of the results From the results
given above, the bread on which ginger extracts
was added was found to be covered with most of
the mould while mould was not found on the other
bread on which wasabi extracts was added. The
similar result was also found in another set of
experiment. Therefore, it can be concluded that
wasabi can inhibit the growth and reproduction of
mould most efficiently. Moreover, the coverage
of mould was found to be higher in the smell-test
of food extracts than that in the solution-test
of food extracts. Thus, it is indicated that the
power to inhibit the growth of mould of the smell
of food extracts is not as strong as that of the
solution of food extracts.
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On the other hand, from the above photo shown,
the colour of mould of the upper row of breads
was different from that of the lower row of
breads. This is due to different kinds of mould
cultivated.
Errors a) The most exasperating problem
confronting attempts to cultivate moulds is
contamination by other moulds. Most mould spores
are very light and easily transported by air.
Even opening the lid of a petri dish for a few
seconds may allow the entry of contaminating
organisms. b) After the pieces of bread (2 cm x
2cm) were cultured with mould, the size of this
could not be maintained. Thus, the size of it not
exactly 2 cm x 2cm. c) Mould did not distribute
equally throughout the bread, thus the amount of
mould on different pieces of bread was not the
same.
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Improvements a) In order to culture pure mould,
a tooth stick was used to take some mould from
the bread cultured with mould onto another bread
and then allow mould growing on the bread. This
can make the mould become more pure.
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b) In order to provide a suitable environment
for the growth of mould, several conditions are
needed to be considered. Suitable
Temperature Some micro-organisms can grow at 50
- 60? others have optimum growth temperatures
of 25 - 40? , and others grow well at 10 - 20?,
and survive at 0? pH value Most
micro-organisms grow best at a pH of 6.6 to 7.5.
Some micro-organisms grow at lower pH values. The
spoilage of fruits is usually caused by yeasts
and moulds, which can tolerate low pH. Moisture
(water activity of the food) The water activity
of a food describes the amount of available water
in a food. It is given by
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Water activity (water vapor pressure above the
food at a certain temperature) / (water vapor
pressure above pure water at the same
temperature) The microorganisms cannot grow with
the water activity less than 0.6. Nutrients to
supply energy, to supply nitrogen for protein
synthesis and minerals Time Some bacteria
divide once every 20 minutes. If the food supply
were adequate, a single bacterium could produce 2
million offspring in 7 hours. The growth rate
declines as the food supply is exhausted A
suitable atmosphere Some microorganisms such as
mould and some bacteria cannot thrive in absence
of oxygen. Yeast and few bacteria can thrive in
either the presence or the absence of oxygen.
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Prevention of contamination The best way to avoid
contamination is by cleanliness. Keep the work
area as clean as possible, wiping down all
surfaces with a commercial bleach solution before
starting to work. The major source of
contaminants in pure cultures is airborne dust.
While cleanliness is essential, it will not
completely stop contamination the cleanest of
laboratories will still have problems. The number
of airborne spores can be reduced drastically by
eliminating as many air currents as possible.
Fans, air-conditioners, and all kinds of traffic
stir up dust continuously and should be avoided.
At least an hour before beginning to work, shut
the windows and doors, turns off fans and
air-conditioners, and stays out of the room. Such
precautions can be effective, even under
difficult field conditions.
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While transferring plate or tube cultures, open
their lids for as short a time as possible. The
less time allowed for entry of foreign spores the
better. It is also wise to avoid opening Petri
dish lids any wider than is needed for the
accomplishment of the work in progress. Spores
sometimes stick to necks of tube cultures and can
fall into the tube. For this reason it is wise to
flame-sterilize the mouths of all tubes and
bottles that one opens. Another design of the
experiment for the same aim 10 g of flour is
put into the mixing bowl with water and yeast to
form bread dough. Then, the bread dough is
divided into 5 smaller one with equal mass and
put into 5 test tubes respectively.
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Glass rod is used to push the bread dough to the
bottom of test tube and flatten the surface of
the bread dough. Then mark the height of the
bread dough. 4 layers of gauze are wrapped the
mouth of the test tubes.
Pepper, Wasabi, Garlic, Ginger are put into 4
prepared test tubes with bread dough. The
remaining one is acted as control.
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After a period of time, measuring the height of
the bread dough again. Explanation of
principal The height of the bread dough that
acts as the control will increase because of the
presence of yeast. If the activity of yeast is
inhibited by the specific food, the height of the
bread dough may not increase as fast as the
control or even stop increasing. From the
experiment, the ability of the specific food to
inhibit the growth of yeast can be tested.
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Background Information What are Fungi?

• Fungi belong to a separate Kingdom to Plants and
  Animals.
• They have no chlorophyll, so they cannot use
  light from the sun to make their own food.
• They don't eat their own food but break down dead
  and decaying plant and animal matter before it is
  absorbed.
• This makes fungi very important in the cycle of
  life.
• There are 5 Kingdoms of organisms
• Plants
• Animals
• Bacteria
• Fungi
• Single cell organisms and algae

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People often think of mushrooms as a type of
plant. Mushrooms belong to a separate group of
organisms called fungi. Other types of fungi
include toadstools, puffballs, truffles, yeast,
and bread mould and skin infections like tinea.
Types of Fungus What types of fungi are there?
If you find a toadstool or mushroom, one way of
identifying what type of fungus it might be, is
to look at its spores. To do this you can make a
spore print. To make a spore print Carefully
cut the stem of fungus from the cap. Place the
cap, with the gills down, on a piece of paper. If
the gills are light coloured, use dark paper. If
the gills are dark, use white paper
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Place a bowl over the fungus to prevent draughts
affecting your spore print. Leave the fungus
for a few hours to allow time for the spores fall
and produce a pattern on the paper. Some fungi
may need to be left overnight, some will work
very quickly. When you have your spore print,
you can keep it by spraying it with a fixative
spray. The fungi kingdom is divided up into
different groups called phyla. Phylum
Basidiomycota These are the most recognizable
fungi they include the mushrooms and toadstools.
They produce their spores on surfaces called
basidia. This is what covers the gills of
mushrooms. Some of the main groups of
basidiomycota are
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Gilled fungi Pore
fungi Coral fungi
Puffballs Spine fungi
Below ground fungi Bracket fungi
Jelly fungi Phylum Ascomycota Fungi
in this group produce spores in tiny sacs called
asci. Some of these are edible, some of these
are Morels
Truffles Yeast
Phylum Zygomycota Moulds
Phylum Chytridiomycota
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Fungi Interactions
What relationships do fungi have with other
organisms? Fungi have a special role to play in
ecosystems. Because they get their food from dead
plants and animals, they are the reason that dead
things decay. Fungi, therefore, are referred to
as decomposers. This allows the food and
nutrients to be returned to the soil and be made
available to other organisms in the ecosystem.
Without fungi we would be covered in piles of
dead plants and animals that have never
decomposed.
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You may notice that many types of toadstools
appear on the ground beneath trees. The mycelium
of these fungi grows in and around the growing
roots of the trees. The fungus is able to get
food from the tree and also allows the tree to
obtain more nutrients, such as phosphorus, from
the soil. This is a beneficial relationship for
both the tree and the fungus. The fungus has a
source of food while the tree can withstand
harsher conditions with a better nutrient supply.
These types of fungi are called mycorrhizae.
Some fungi can be parasites or cause diseases.
They may infect plants or animals and sometimes
cause their deaths. People can also get fungal
infections. Tinea and ringworm are both
infections of types of fungus.
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Fungi Close-up
What is a fungus made up of? Some people think
that toadstool or mushroom is the main part of
the fungus but this is really just the
reproductive part the main part of the fungus is
under ground. This main part is called the
mycelium and is made up of very fine tubes called
hyphae. The hyphae are only one cell wide so that
is easy for each cell to obtain food. What does
the mushroom part of the fungus do? The mushroom
or toadstool part of the fungus is the fruiting
body and is used for reproduction. Spores are
produced in the gills of the mushroom. Air
currents disperse these tiny spores. When they
land in a suitable place, they germinate and grow
into new hyphae.
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MOULDS AND THEIR CHARACTERISTICS Moulds, those
dusty little spots often found spreading over
bread, cheese, books, and other things in the
home, cause the loss of millions of dollars to
our economy every year and, even worse, may be a
menace to your health. To deal with them
successfully we must understand what moulds are
and exactly what they are doing. Moulds are
microscopic, plant-like organisms, composed of
long filaments called hyphae. Mould hyphae grow
over the surface and inside nearly all substances
of plant or animal origin. Because of their
filamentous construction and consistent lack of
chlorophyll they are considered by most
biologists to be separate from the plant kingdom
and members of the kingdom of fungi.
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They are related to the familiar mushrooms and
toadstools, differing only in not having their
filaments united into large fruiting structures.
When mould hyphae are numerous enough to be
seen by the naked eye they form a cottony mass
called a mycelium. It is the hyphae and resulting
mycelia that invade things in our homes and cause
them to decay. Moulds reproduce by spores they
germinate to produce a new mould colony when they
land in a suitable place. Unlike seeds, they are
very simple in structure and never contain an
embryo or any sort of preformed offspring. Spores
are produced in a variety of ways and occur in a
bewildering array of shapes and sizes. In spite
of this diversity, spores are quite constant in
shape, size, colour and form for any given mould,
and are thus very useful for mould
identification.
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The most basic difference between spores lies in
their method of initiation, which can be either
sexual or asexual. Sexually initiated spores
result from a mating between two different
organisms or hyphae, whereas asexual spores
result from a simple internal division or
external modification of an individual hypha. The
recognition of a mating and subsequent spore
formation is often difficult for an observer, and
is usually reserved for patient specialists.
However, for practical purposes one can learn to
recognize certain indications of the sexual
process, namely, the four kinds of sexually
determined spores that appear in mould fungi
(1) Oospores, (2) Zygospores, (3) Ascospores,
and (4) Basidiospores
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Common mould on bread
Rhizopus stolonifer
Penicillin expansum
Aspergillus niger
Chrysonilia sitophila
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Other kinds are Head mould (mucor) on fruit and
bread. Flask (spadix) or pouring jugs
(Aspergillus), on moist plant matter, fruit,
bread, and leather. Brush mould (botrytis
cinera), perishable fruit, especially
strawberries and half ripening grapes. Other
kinds of moulds serve as the basis for
Antibiotics. Here we shall just mention
penicillin and streptomycin. First mould fungi
(and yeast fungi) show activation when radiated
with small doses of UV. With larger doses they
are killed, in which process the spectral effect
is approximately the same as with the bacteria.
Also here the spores are essentially more
resistant than vegetative kinds.
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• Other alternatives of the experiment
• 1) Preparation of mould
• Moulds are microscopic plants. They are not like
  green plants, which make their own food from
  light. Moulds actually eat other things. It grows
  on cheese, oranges bread, and other items that
  can be found in home.
• Try this experiment and make mould grow.
• Materials
• 2 cups of flour
• 1 cup of salt
• 1 tablespoon of oil
• 3/4 cup water
• white bread (no crust)
• measuring cups
• measuring spoons
• a mixing bowl
• a big spoon
• 2 jars with lids

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• Procedure
• Part 1
• Mix the flour, salt, oil, and water together in
  the mixing bowl. Mix until it reaches the
  consistency of bread dough.
• The mixture will dry very quickly. Keep it in a
  covered jar or plastic bag when you are not using
  it.
• Part 2
• Rub your hands in some soil to get them very
  dirty.
• Handle the bread and the dough thoroughly.
• Put the bread and the dough in separate jars.
  Screw the lids tightly on the jars. Label them
  Bread and Dough. Place the jars near a window.
• After several days observe what has happened in
  each jar. Mould should be growing on the plain
  bread, but not on the oil-and-salt dough. Why is
  this so? What are some ways the bread and dough
  are different?

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What happened?
Mould grew on bread dough! The bread dough and
salt provided food for the mould. Where did the
mould come from? Mould spores (like seeds) are
everywhere. They are even flying around in the
air. More mould probably grew on the bread dough
with the dirt on it. The reason why more mould
grew on the bread dough with dirt because dirt
acts like glue, and catches mould spores. There
were more mould spores on the dirty bread dough
than the clean bread dough, so more mould grew on
the dirty bread dough.
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• Why did you have to seal the lids on the jar?
  Mould likes moisture. By closing the lid of the
  jar, the moisture gets trapped inside the jar
  providing the perfect environment for the mould
  to grow.
• (2) Comparison of the effect of different fluids
  on the percentage of the bread that is covered by
  the mould
• Label one zippered plastic bag as "Dry White
  Bread", a second as "Water on White Bread", a
  third as "Lemon Juice on White Bread", a fourth
  as "Sugar Water on White bread"
• Place a slice of dry white bread in the bag
  marked "Dry White Bread" and seal.
• Sprinkle 20 drops of water on another slice of
  white bread. Don't over-moisten the bread. It
  should be damp - not wet. Place it in the bag
  marked "Water on White Bread" and seal.

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• Sprinkle 20 drops of lemon juice on another slice
  of white bread. Place it in the bag marked "Lemon
  Juice on White Bread" and seal.
• Sprinkle 20 drops of sugar water on the last
  slice of white bread. Place it in the bag marked
  "Sugar Water on White Bread" and seal.
• Repeat the steps above, but use a different type
  of bread in the other four bags.
• Label all the bags.
• Place all 8 bags in a dark, warm place (the same
  for all the bags).
• Wash your hands, and be very careful not to
  inhale the mould spores
• Check the bags daily for two weeks. Record your
  results (write a brief description and draw the
  mould). Graph paper is a good way of showing how
  much the mould spreads.

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If you have access to a microscope or if you have
a magnifying glass, have a closer look at what
mould looks like. Work out the percentage of the
bread that is covered by the mould. Construct a
graph like the one below - but with your real
results
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Further Discussion Fungi contain different
nutrious chemicals in which some of them can be
used for medicine. Also, according to some
biological research, some of the sorts of fungi
can help prevent cancer as well. But it may cause
food spoilage. And, mould can cause illness,
especially if the person is allergic to molds.
Usually though, the main symptoms from eating
mouldy food will be nausea or vomiting from the
bad taste and smell of the mouldy food.
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Garlic and Cancer There is now a lot of
information published on the role of garlic in
cancer. Recently, researchers in Pennsylvannia
have shown that by injecting a compound called
diallyl disulphide (formed when raw garlic is cut
or crushed) that tumours can be reduced by half
and that a further compound (S-Allylcysteine) can
stop cancer causing agents from binding to human
breast cells. The search for compounds that
prevent cancer has intensified with the mounting
evidence that many types of cancer are caused or
triggered by factors relating to lifestyle and
environment. It is well known that garlic can
strengthen the immune system, which is vitally
important for fighting cancer.
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Recent findings support a growing body of
evidence that garlic works as an anti-carcinogen
in both prevention and treatment, and that garlic
and related foods play an important dietary role
in the cancer process. For instance, scientists
have correlated garlic intake with reduced
nitrite levels in people and fewer deaths from
stomach cancer are recorded. Currently it is
unknown specifically how garlic protects but it
may relate to blocking formation of
cancer-causing compounds, stopping their ability
to form tumours or even inhibiting the growth of
tumour cells. This follows on from work
conducted by Professor Michael Wargovich at the
University of Texas where his group studied the
effects of two major purified components of
garlic - dialylsulphide, which is soluble in oil,
and S-allyl cysteine, which is water-soluble.
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He tested these compounds on two animal carcinoma
models and found that the tumours could be
reduced by between 50 and 75 Then in his
controls ( used to remove any experimental bias)
he gave a prophylactic dose of garlic and then
deliberately tried to induce a particularly
virulent form of oesophageal cancer. He found
that garlic completely prevented his experimental
controls from becoming infected. His conclusion
was that although the precise mechanism may not
be clear his results had shown that the
administration of well-tolerated garlic products
may confer important protection from cancer.
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The End