Exercise A Investigating Herbal Remedies With the Shrimp Bioassay

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A bioassay uses a living organism to test the toxicity of chemicals. One of the earliest bioassays was employed by miners who worked in underground coal mines. One by-product of coal mining is the release of methane, a deadly, odorless gas Caged canaries would accompany the miners on their journey deep into the earth to act as a bioassay for the presence of methane. If the birds died, methane concentrations in die mine were at dangerous levels. a warning that the miners should evacuate.

You will be using a bioassay to detect for bioactivit) in plant extracts. The rauonalc behind the bioassay is that if the active ingredient in a plant can impair herbivores, as shown by killing off brine shrimp, perhaps it can be used to kill off cancer cells or to inhibit the spread of an infection. Also, since the classification of a plant principle as toxic versus therapeutic is often a matter of dosage, any evidence of lethality to the test organisms can indicate that the plant has the potential to act as a natural medicine at a lower concentration.

Many different organisms have been used in laboratory bioassays. The water ilea (Daphnia) and wrigglers (mosquito larvae) are two common examples, but you will be working with an equally well-known organism of choice, the brine shrimp.

Brine shrimp. Arte win saltan, arc microscopic crustaceans (fig. 16.1). Commonly known as sea monkeys. they thrive in extremely salty environments, such as the Great Salt Lake and ponds at saltworks. The life cycle of a brine shrimp is about a year long. Newly hatched eggs produce larvae, or nauplii, that go through 15 molts before reaching the size (13 mm in length) of sexually mature males and females. Brine shrimp are filter feeders, feeding on bacteria and algae that can also tolerate the highly saline conditions of their environment. When environmental conditions arc unfavorable—for example, when a pond dries out—fertilized eggs encyst (become covered with protective cases). These cysts are extremely durable and remain dormant until conditions are favorable again.

You will perform the brine shrimp bioassay on selected healing herbs. Some suggestions of herbs to test are listed in table 16.1, or you may bring in a plant you have collected from die wild or bought from an alternative health store

Brine Shrimp Bioassay


Materials Needed

Aquarium pump

Beakers, 50 ml

Brine shrimp

Dissecting microscopc

Gang valve, 4 way

Graduated pipet, 5 ml. or 10 ml graduated cylinder

Marker pen

Medicine droppers

Methanol, 100%

for Exercise A

Micropipctors Mortar and pestle Pasteur pipets Petri dishes Pipet pumps Plastic tubing Sea water Template vial Test tube rack Vials, 2-dram

Procedure for Exercise A

  1. Organize into research teams of four students. Select an herbal preparation to investigate from table 16.1. or an herb of your own choosing. Using a mortar and pestle that is clean and dry, grind the dried plant material into a powder, the finer the better. Weigh out 0. Ig of the powder, and placc it in a small (50 ml) beaker. Add 10 ml of 100% methanol to die plant powder. Let this set for 25 minutes. The methanol is a solvent that should extract any bioactivc compounds from the plant material.
  2. While the mcdianol is dissolving any bioactivc compounds in the plant material, set up the air lines (fig. 16.2 i. Connect one end of the air line to the aquarium pump and the other end to a gang valve. From the gang valve, connect four air lines and a Pasteur pipet at the end of each line. These air lines will be used to evaporate the methanol from the plant extract. It is essential diat the methanol Ik* completely
Brine Shrimp Bioassay Procedure










Calendula officinalis



Combats fungal, bacterial, and viral infections

Echinocca purpurea

Purple coneflower


Helps fight off flu and colds

Hydrastis canadensis



Antisepac/anobiotic, Fights infections

Panax ginseng



Speeds recovery from illness

Camtllia vnertss

Green tea

Unfermented leaves

Inhibits many cancers

Voconium oxycoccos



Clears up infections

Valeriana officinalis




evaporated from the extract because methanol in itself would be toxic to the brine shrimp.

  1. Set up and label the vials or test tubes for this experiment. Kach test will require 10 vials. Label a set of live vials 1A-5A and label a second set IB-5B. Vials 1A and 1B arc the seawatcr controls. Only seawater will be added to these vials, as described in step 8. No plant extract will be added to vials 1A and I B. Vials 2A and 2B arc the methanol controls. You will add SO u\
  2. of 100% methanol to each of these vials. No plant extract will Ik added to vials 2A and 2B.
  3. Tubes 3A-5A and 3B-5B are the experimental vials. These will contain three concentrations of the plant extract. To vials 3A ami 3B, you will add 5 /il of the plant extract. To vials 4A and 4B. you will add 50 /il of the plant extract, and to vials 5A-5B you will add 500 u\ of the plant extract.

With the addition of a final volume of seawatcr i described in step 8), the concentrations of the extract tested will be 10 /ig/ml (vials 3A. 3B>, 100 /ig/ml (vials 4A, 4B.. and 1,000 /ig/ml (vials 5A, 5B). The experimental protocol is outlined in table 16.2.

  1. Two members of die research team have the task of counting out the brine shrimp. Ten brine shrimp will Ix: added to each vial. Obtain a small sample of water from the brine shrimp aquarium. Each person doing the counting should have a dissecting microscope, a petri dish, and a medicine dropper. Create a small pud die of brine shrimp on one of your pctri dish lids. Using the mcdicinc dropper, count out 10 brine shrimp and move them to a new puddle in another petri dish lid. Make sure no cysts are included. l"hc cysts are sphcri cal and golden-brown in color. Also, make sure the puddle contains enough seawatcr to keep the brine shrimp from doing out before they arc placed in one of the vials. Add to die puddle as need be from the pure seawater. Repeat this procedure until you have 10 pud dies, each with a count of 10 brine shrimp.
  2. Obtain a vial template that has a marker line at the 5 ml measure. Using this template and a marking pen, mark off the 5 ml mark for each of your 10 \ials (1A-5A and 1 B-5B series). The level line will be used later to make sure each vial contains a total volume of 5 ml of seawatcr.
  3. After 20 minutes have elapsed, measure out with a micropipctor the volumes of plant extract as outlined in step 3 and table 16.2 to vial 3A-5A and 3B-5B. Measure out 50 /il (step 3 and table 16.2) of methanol each to vials 2 A and 2B. Start the evaporative process by plugging in the aquarium pump and putting 4 air lines with pipets in test tubes 5A, 5fi, 4A, and 4B. These test tubes contain the greatest volumes of methanol and will take the longest to be evaporated off, so they must be started first. Make sure the toxic methanol is completely evaporated from the extract. Expect a 30-60 minute drying time for vials 5A and 5B. As these extracts are evaporated completely, move to the next set of vials, but make sure you change the pipct with each new vial to avoid contamination The other vials will take much less time. In fact, you may find that as you progress down the concentration line, the vials with smaller quantities of methanol have completely air-dried and will not require the air line at all.
  4. Once the extracts and the methanol control vials have been completely dried, use the pipct pump to measure out and add 2 ml of plain seawatcr to each vial including lubes 1A and IB. Then add .1 puddle of 10 brine shrimp to each vial. Now bring up the total volume in each vial by adding seawater to the level of the 5 nil line.
  5. Label the test tube rack with die date, rime, lab section, plant tested, and name of the research group members. Placc the rack in a safe area away irom hot lamps and direct sunlight.
  6. After 24 hours, assess the bioassay. Empty the contents of single vial into a petri dish lid. Rinse out the




1A and 1B (seawater controls)

None, only seawater


2A and 2B (methanol controls)

50 pi of methanol only


3A and 3B

5 Ml


4A and 4B

50 pi


5A and 5B

500 pi


vial with a small amount of plain scawatcr and also dump this into the pctri dish lid. Count the living brine shrimp using the dissecting microscope and record the numbers in worksheet 16.1. Also calculate and record the percent survivorship for each vial and the mean for each treatment.

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