APES: Use of Brine Shrimp Assay to determine LC-50 of Herbal Teas

The use of herbal products as medicinal remedies has increased greatly in the past decade. We are inundated with advertisements that tell us that tell us the natural remedies will accomplish cures and imply that the term “natural” means free of side effects. Many herbal produces may do what they claim and some do not. Any product that has a desired effect is also likely to have some undesired side effects. Unfortunately, only a few of the herbal remedies have scientific data to back up their clinical benefits, and some are known to be dangerous. Herbal teas remain an under-investigated group of plant products.

Tea used: ______

Purported effects of tea: ______

______

Procedure:

  1. Preparation of tea extract: A cup of tea contains 200 ml of water per teabag (1.0x dosage). You will prepare a 10x dosage by using 4 teabags in 80ml of brine (seawater).
  2. Place 4 teabags flatly onto the bottom of a 100 ml beak
  3. Place 80 ml of hot artificial seawater (brine) into the beaker and let it steep for 15 minutes, shaking gently every 5 minutes. In the meantime, label 24 Dixie cups with the following labels: 10x, 7.5x, 5x, 2.5x, 1x, 0.5x, 0.1x, 0x. You should have 3 cups of each label. Example: label 3 cups 10x, 3 cups 7.5x, etc.
  4. Squeeze each teabag between two spoons. This solution is designated as the tea extract at a 10x solution.
  5. Go to number 2 in the directions to make the different serial dilutions.
  6. Making tea solutions: You will make 3 cups of each of the following solutions: 10x, 7.5x, 5x, 2.5x, 1x, 0.5x, 0.1x as well as a control of 0x. Follow the directions below to make the solutions.
  7. Take a new 50ml beaker. Add 2 ml of initial/dark tea (10x) solution to 18ml of seawater (heated water). The resulting 20ml of solution will be medium color (1x).
  8. Take the medium(1x) solution and take a new 50 ml beaker. Add 2 ml of the medium (1x) solution and 18 ml of seawater to a beaker. The resulting 20 ml of solution will be light (0.1x).
  9. Pour 5 ml of the dark (10x) solution into your cups labeled 10x.
  10. Pour 5 ml of the medium (1x) solution into your cups labeled 1x.
  11. Pour 5 ml of the light (0.1x) solution into your cups labeled 0.1x.
  12. To make the 7.5 solution, place 3.75ml of the dark (10x) solution and 1.25ml of seawater into a cup labeled 7.5x. Repeat twice.
  13. To make the 5x solution, place 2.5 ml of the dark (10x) solution and 2.5 ml of seawater into a cup labeled 5x. Repeat twice.
  14. To make the 2.5x solution, make a 5x solution (in another beaker). Take 2.5ml of the 5x and 2.5 ml of seawater and place into a cup labeled 2.5x. Repeat twice.
  15. To make the 0.5x solution, place 2.5ml of the medium (1x) solution and 2.5 ml of seawater into a cup labeled 0.5x. Repeat twice.
  16. Place 5 ml of seawater into the cup labeled 0x. Repeat twice.
  17. Preparing the shrimp: In this lab, we will use a small crustacean, the brine shrimp. It is normally found in brackish water and is a very hearty little organisms and able to tolerate high salt concentrations. Using a spoon or pipette, transfer 10 shrimp to each cup.
  18. After 24 hours, count the surviving brine shrimp and fill in the attached table.

Data Table: (cut out and place in your field notebook!)

Concentration: / #dead / %mortality / Concentration / #dead / %mortality
10x / 1x
10x / 1x
10x / 1x
7.5x / 0.5x
7.5x / 0.5x
7.5x / 0.5x
5x / 0.1x
5x / 0.1x
5x / 0.1x
2.5x / 0x
2.5x / 0x
2.5x / 0x

Graph: Plot a scatter graph of your data using different colors for the different serial solutions. Place concentration on the X-axis and mortality on the Y-axis.

Analysis Questions (answer in your field notebook!):

  1. Although Brine Shrimp are hardy enough to withstand a wide range of salt concentrations, they are short-lived. Do you have any evidence of a background death rate independent of the addition of herbal teas? Explain.
  2. What is the LC-50 for your tea on brine shrimp?
  3. Based on your data in this lab, what is the safe concentration for brine shrimp? (Lowest observable Effect Concentration?)
  4. If you were to pursue this investigation further (repeat/verify results), what would you do to improve upon this lab?