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(Pictures of possible results
at bottom of page)
Introduction:
A hay infusion is an
ecosystem
containing simple plants and animals. Our population of
interest in this exercise is a population of single-celled
animals (a.k.a. "protists"). The health of the population and
its environment is reflected in the diversity (number of
species) of protists as well as the number of protists supported
in the environment. Here we will see how increasing amounts of
controlled substances: alcohol and tobacco, affect the growth of
the animal population and their diversity. Feel free to test
with other substances as well such as toothpaste, soda,
mouthwash, etc.
Remember to record your observations at each
step!
Procedure:
1. Make an
infusion of hay or dry grass or pond mud.
Combine 100 mLs water with one
tenth volume hay or dry grass (10ml
or 10cm3)or one 100th volume
(1ml) of pond mud in a glass jar. Agitate (mix) gently.
Leave the lid off or loose and incubate at room temperature with
partial sunlight exposure. Agitate the solution every day and
incubate for three days (over the weekend?). Grade
opacity (cloudiness) after large particles settle by trying
to
read fine newsprint through the solution.
2. Perform treatments
with ethanol and tobacco.
On the third day, agitate the
infusion and allow large particles to settle for 5 min. Score
the opacity of the upper solution. Take 12 - 5 ml (1 teaspoon)
samples from the top of the infusion and put them into glass
test tubes.
| Tube |
Infusion mix |
Water |
Ethanol |
Tobacco |
| 1C |
5ml |
1drop |
X
|
X
|
| 2C |
5ml |
2drops |
X
|
X
|
| 3C |
5ml |
4drops |
X
|
X
|
| 4C |
5ml |
8drops |
X
|
X
|
| 1E |
5ml |
X
|
1drop |
X
|
| 2E |
5ml |
X
|
2drops |
X
|
| 3E |
5ml |
X
|
4drops |
X
|
| 4E |
5ml |
X
|
8drops |
X
|
| 1T |
5ml |
X
|
X
|
1mm2 |
| 2T |
5ml |
X
|
X
|
2mm2 |
| 3T |
5ml |
X
|
X
|
4mm2 |
| 4T |
5ml |
X
|
X
|
8mm2 |
-
Label tubes 1C, 2C, 3C, 4C,
1E, 2E, 3E, 4E, 1T, 2T, 3T, 4T (12 tubes total.)
-
Using an eyedropper, add 1,
2, 4, or 8 drops of water into tubes 1C, 2C, 3C, and 4C,
respectively. Add the same amounts of ethanol (alcohol) to
tubes 1E to 4E, respectively. Add increasing amounts of
tobacco to tubes 1T to 4T so that each has twice as much as
the preceding tube. Start with a very small piece of tobacco
(about 1 mm2) for the low dose because it is VERY
POTENT!
-
Mix gently by sealing the top
with Saran wrap and inverting the tube while holding the
Saran wrap in place.
* (feel free to experiment with other substances such as
toothpaste, coke, etc)
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3. Determine immediate
effects of treatment:
Put a drop of culture on a glass
slide and apply a cover slip. Using a microscope, count and
characterize the moving microfauna (protists). You may see
single-cell animals such as Euglena, Paramecia, Amoebae, and
rotifers (see pictures below.) Most should move purposefully,
that is, go places (some very fast!) and react to encountering
obstacles. Distinguish this from flowing solution effects where
everything is moving at the same speed and direction.
To compare treatments: The lowest
doses of ethanol and tobacco in 1E and 1T should be compared to
the 1C control tube (untreated). Compare higher doses in 2E - 4E
and 2T - 4T to 2C-4C.
Record you results on the Treatment effects observation pages.
4. Determine the
effect of treatment over time on the growth and health
of the infusion protists.
Incubate infusions in partial
sunlight. Agitate, settle and grade opacity once daily for two
days. Assess number of animal cells and species diversity as in
#3
above. What are the trends with increasing alcohol and
tobacco?
5. Prepare DNA from the
infusions.*
Another way to quantify cell
number is to prepare DNA from the cells. To analyze the effects
of treatments on growth of infusions:
This is DNA spooling of
the chromosomal DNA molecules. Faster and larger
spooling indicates more live cells were present in the
original infusion. Smaller, slower spooling indicates
inhibition of growth and/or death of cells followed by
destruction of the DNA.
Do this for treated and control
infusions. Make the same comparisons described in #4 to
assess differences to controls and dose effects of alcohol
treatment.
1) Do the DNA results correlate with the
opaqueness of the infusions?
(Record your
observations and send them to us!)
*DNA precipitation (spooling) requires a
minimal DNA concentration. Thus the most turbid (cloudy)
infusions yield the best results.
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TEACHER NOTES:
1. THIS EXERCISE CAN BE
STREAMLINED TO TAKE ONLY 45 MIN OF CLASS TIME. It just
takes prior preparation of the infusions 5 days before
they are needed (step 1) and administration of
treatments the third day of infusion growth (step 2).
Save stock infusion for student to do high dose
treatments only along with controls (3 tubes total: "E"
= 8 drops ethanol, "C" control = 8 drops water and "T" =
8mm2 tobacco). The students can look at these
tubes under the microscope to record observations of
immediate treatment effects on cells (step 3). They can
use all or a subset of the infusions treated 2 days
previously to do the DNA isolation in steps 4 and 5.
2. In some cases we have seen more
growth in cultures with tobacco, from protozoa able to
use it as food. In this case, students could assess the
diversity of the protozoa as an indication of ecological
health. Since only a subset of protozoa can use the
tobacco, they may see less diversity even though numbers
of protozoa cells are greater. It is like a wild species
overpopulation in an unbalanced environment.
3. A tobacco infusion can be used to
replace the small flakes of tobacco. Soak 1oz of tobacco
in 1/2 cup of water overnight. Then use drops of tobacco
infusion instead of mm2 (1, 2, 4, 8.)
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Experiment designed by Dr. Nancy Ing, Texas A&M
University
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Click
here for pictures of possible results
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