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This activity is designed to get students to understand that alcohol and tobacco are toxic to living cells. Study the instructions below. Complete the experiment, recording observations on the student journal activity sheet.

For a streamlined procedure, see Teacher's Note #1 at end.

Supplies: see below

Access the Student Journal for Activity 2

Materials Needed :  

Hay, dry grass or mud from the edge of a pond

(1) glass jar, glass rod

Microscope, glass slides, and cover slips

Fine newsprint

(36) test tubes (8ml or more)

Grain alcohol (100% ethanol) - needs to be ice cold for part B: DNA extraction

Saran wrap

Tobacco (see Teacher's Note 3 at end)

An eyedropper

Saran wrap

Shampoo

Salt solution (Fill small container 1/4 full of salt. Add water and shake 2 min. After the mixture has settled, undissolved salt is on the bottom.  Use the saturated solution above it.

Activity #2 - Ethanol and Tobacco Toxicity in Living Animal Cells

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.

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 towpeD.gif (20404 bytes) 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.

 

 

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.

Aprotists3.jpg (16041 bytes)

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:

  • Agitate then allow the infusion particles (plant materials used for food) to settle 1 min. After settling, take out 10 drops of infusion (containing live protozoa) from the bottom of the culture tube and place it in a new tube.

  • Break the cells by adding 1 drop of hair shampoo, mixing the solution well and letting it sit in its tube in a cup of hot tap water for 15 minutes (water should be about 65o C). The solution should become clear as particles (cells) dissolve. Finally, cool the solution in ice water.

  • DNA requires salt and alcohol to precipitate. Add 3 drops of saturated salt solution and mix. Then add 1 ml (20 drops) of ice-cold ethanol. The froth will disappear. IMPORTANT: Observe but DO NOT MIX at this point. DNA strands will precipitate as very fine, almost clear, white threads that form a ball of unorganized strands.

  • Stick a glass rod (needs to be glass) into the upper part of the solution and slowly stir, the DNA strands will wind together to form a "spool". This spooling will allow you to pick up the DNA on the glass rod. It will look like a wet, whitish glob. (The DNA is now purified away from RNA and protein macromolecules. You could transfer the "glob" to a clean tube, dissolve it in water and subject it to an experiment like DNA sequencing.

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.

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.)

Experiment designed by Dr. Nancy Ing, Texas A&M University

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