Experiment
Design:
Design
an experiment to test each hypothesis. Make a
step-by-step list of what you will do to answer
each question. This list is called an experimental
procedure. For an experiment to give answers you
can trust, it must have a "control." A
control is an additional experimental trial or
run. It is a separate experiment, done exactly
like the others. The only difference is that no
experimental variables are changed. A control is a
neutral "reference point" for comparison
that allows you to see what changing a variable
does by comparing it to not changing anything.
Dependable controls are sometimes very hard to
develop. They can be the hardest part of a
project. Without a control you cannot be sure that
changing the variable causes your observations. A
series of experiments that includes a control is
called a "controlled experiment."
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you will see how to grow bacteria for the
purpose of counting bacteria.
You need
to use a diluted solution and grow it in a
nutrient agar plate. Record the time.
After 36 hours or 24 hours each bacteria
will become a colony. Use a sterile object
to remove one spot or one colony and
perform bacteria count test on it. For
bacteria count you need to dilute this
sample and grow it again on another set of
dishes. This will show you how many
bacteria are produced from one bacterium
in 24 or 36 hours.
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In order to grow
bacteria, you will need culture media, plates or
petri-dishes and some laboratory supplies and
incubator.
Culture
Media: Culture media is a moist or liquid
matter that contains nutrients for bacteria.
Almost any nutrient food may be considered a
culture media for general bacteria, however if you
want to grow a specific bacteria or prevent
growing some other bacteria, you will need to use
a fine tuned recipe for your culture media.
Chicken broth
and beef broth are among nutrients that most
bacteria like. In some recipes you may also add
some mushroom extract. Sugar can also be added to
most culture media. Small amounts of some minerals
such as potassium phosphate and calcium carbonate
may also be added to the culture media. Note that
there are many foods that are good for growing
bacteria, but they are not good as culture media.
For example bacteria can easily grow on milk, but
milk is not a good culture media because it will
change by the activity of bacteria. Part of milk
will solidifies when bacteria produce acids. A
good culture media must be clear and must remain
liquid and should not easily change pH. If we need
to solidify our culture media, we use agar to do
that. Agar is a gelatinous substance that is
extracted from sea weeds. If we need to grow
bacteria for the purpose of identification or
counting, we need to grow bacteria in nutrient
agar plates. These are petri-dishes containing a
mixture of agar and nutrients.
Peteri-dishes:
Petri-dishes are disposable clear plastic dishes
with a cap that are used for many science
experiments and bacteria growth. A thin
layer of nutrient agar in a petri-dish is enough
for growing bacteria. You can see the bacteria
colony shapes and count them without opening the
lead of the petri-dish. Since petri-dishes are
clear, you can see the bacteria from either side
of the dish.
Incubator:
Incubator is a warm cabinet that you can set it's
temperature to a proper temperature for bacteria
growth. About 35º C is a good temperature for
most bacteria. This is close the body temperature.
If you be able to create such a temperature in any
other way, it is as good as an incubator. You may
find warm places behind your refrigerator, next to
the radiator or inside an oven that is off.
You may also
make an incubator by placing a small desk lamp
inside a wooden or metal box. Or you may put a
Styrofoam cooler upside down over a desk lamp. A
small lamp (15 watts) should be able to create
enough heat to warm up a small space. Prepare your
incubator in advance and use a thermometer to test
it a day before starting your experiment.
Material:
- Agar (dry
powder)*
- 10 petri-dishes
(100 mmx 15mm)*
- 1 ml Pipette*
- 10 ml pipette*
- 2 transfer
pipette*
- 2 test tubes
with cap*
- Glass beaker
or steel pan *
- Chicken broth
or beef broth *
- Filter Paper *
*
These material are included in a kit
from MiniScience.com or you may buy them
individually from a local laboratory supplier.
*
Chicken broth or beef broth can be purchased from
supermarkets and health food stores or you may
make them at home. (It must be fat free). Filter
paper is coffee filter or you may substitute it
with any clean cotton cloth.
Procedure:
If you are
making your own chicken broth, one small chicken
can give you enough broth for this experiment.
(Half a pound beef can be used instead). Boil the
chicken for one hour. Separate the broth by
transferring it to another pot. remove any fat
from the top of the broth. Filter the broth using
a clean piece of cloth or coffee filter. If you
are buying chicken or beef broth from a super
market, it comes in small bags of about 4 grams
each. Use two bags in about 300 ml water and boil
it. Then filter it and transfer the filtered broth
to another clean pan.
Add water to the
broth to bring it to about 650 ml and boil it
again. Start adding the Agar powder while
stirring. (If you are using a MiniScience
kit, entire agar tube must be used, otherwise use
8 grams of dry agar powder). You will add agar
gradually while stirring. Adding agar will take 30
seconds to one minute. If you don't stir it good,
agar solidifies at the bottom of the pan and
burns. Continue stirring for another two minutes
or until the agar solution is fully dissolved and
the solution is clear.
At this time you
may optionally add a calcium carbonate tablet and
some mushroom extract. (You could also boil
mushroom with beef or chicken broth).
Your nutrient
agar is ready at this time. Turn off the heat and
let it cool down. While it is still warm set your
petri-dishes on a table. Open the lead of each
petri dish and pour some nutrient agar in each
dish (enough to cover the bottom of the dish) and
immediately put the lead back. Repeat this for all
petri-dishes. Leave the dishes where they are
until they solidify.
Note: The
nutrient agar should be free from all bacteria,
however boiling usually is not enough to kill all
the environmental bacteria that have entered our
nutrient agar. For school experiments, it may not
matter but in real biology laboratories, nutrient
agar plates are transferred to an autoclave for
sterilization. Hot, under pressure steam of
autoclave can kill all bacteria in about 30
minutes. Advanced students may use a pressure
cooker to do the same.
Prepare your
test sample:
For counting
bacteria, you will prepare different dilutions of
your original sample. For example you will make
1:10, 1:100, 1:1000 and 1:10000 dilutions. You
will then grow each dilution on a different
nutrient agar plate.
Use a 1 ml
pipette to place 0.1 ml of each dilution in each
dish. Spread the liquid all over the plate surface
using a sterile spoon. Cover the plate. Turn it
upside down and place it in an incubator. Nutrient
agar will remain moist when the plate is upside
down. Check back within 36 hours to see the result
of bacteria growth.
When the bacteria
grow, each bacteria will become a bacteria colony
that can be seen as a small spot on the petri
dish. By counting bacteria colonies, you will know
how many bacteria existed in the sample.
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