Protocol to use the brine shrimp assay to test the potency of medicinal plant extracts
author: Dr. Kevin Curran
We have been using this assay in the USD Biology Dept.
- Collect plants and create extracts.
- Grow your brine shrimp.
- Incubate hatched brine shrimp in plant extract.
- Count mortality rates after 24 hours incubation.
Now for some details…
Protocol for performing methanol extraction of medicinal plants
- Break up plant matter, dry in sun on clean surface or in desiccator.
- Prepare 70% methanol. Use large graduated cylinder (700ml of 100%methanol/300ml water).
- Weigh out 10 grams of dried plant material from each sample. Chop or tear up plant material to provide greater surface area for extraction (about 1/4 inch sized pieces)
- Move dried plant material into 250 ml. graduated cylinders.
- Add 200 ml of 70% Methanol.
- Label tubes (plant name, operator initials, date and time of beginning of extraction.)
- Allow to extract for 3-4 days. Stir often – either manually or on rotating platform.
- Fold large Whatcom filter in a cone. Place coned filter inside plastic funnel. Put piece of tape on filter to attach to filter.
- Pour methanol extraction into filter cone. Allow extract to drip through.
- Squeeze out final methanol trapped in plant material. Do this by removing wet plant material from drained bottle. Move material into coffee filter. Hold coffee filter over filter cone and squeeze gently. This is important to capture compounds trapped in material.
- Store extract in Freezer (-20°F), ideally in a brown, tinted bottle. In general, compounds should store well (not degrade) for a 6-8 month time frame in -20°F. At refrigerator (4 °F) you can expect half of that. Of course, every compound will be slightly different but these are general conditions for a broad collection of compounds.
Protocol for hatching the brine shrimp larvae
Combine water, sea salt and brine shrimp larvae (BSL) in tank. BSL should be floating in the tank for 24 hrs (hatching) before testing with extracts begin.
- Combine 470 ml of water with 1 tbsp of Instant Sea Salt (for marine aquariums)
- Mix thoroughly in large plastic cylinder using parafilm.
- Pour into plastic tank (empty coke bottle).
- Add in ½ teaspoon of BSL (1 gram weight)
- Turn on the bubbler (cheap aerators can be found at Pet Stores).
- Place lid on top of tank.
- Return the following day to harvest your larvae.
- Hatching is optimal at 70-80 ° F.
Protocol to test BSL (brine shrimp larvae) in 24 well trays to test plant extracts
- Label a 15 ml. conical tube for each test condition.
- Perform a C1 x V1 = C2 x V2 equation to determine the volume of plant extract in 70% ME (Methanol) to add to 15 ml conical tube and to determine volume of sea water necessary to bring conical tubes up to final volume.
- Pipette determined volume of plant extract (70% ME) into 15 ml conical.
- Before bringing test solution to final volume, add in ~50 BSL. These will be in sea water, which is good. Number of duplicate wells being tested will determine approximate number of BSL. For example, if you are filling 3 wells, then you need 30 BSL total. Therefore, 50 is a safe number to use for 3 wells.
- Bring final volume of 15 ml conical tube up to the 15 ml. You now have about 50 BSL swimming in 15 ml of your test condition at the correct concentration. The 24 hour period of exposure has officially begun at this moment.
- Label the lid of the 24 well tray. Write with lab marker on lid, so it is clear which wells are getting which test condition.
- Move 10 BSL into each well from appropriate 15 ml conical tube. This can be done by pouring the test condition/BSL into a shallow dish. With plastic pipette shuttle 10 BSL into each well. You may find it easier to pipette up 10 BSl with the unaided eye (as opposed to using a dissecting microscope).
- Allow the BSL to swim in test condition for 24 hours total.
- After incubation period, return and count % lethality.
The internal volume of each well in a 24 well plates is 3 ml. We fill each well with 2 ml of extract/larvae.
The final concentration of extract and methanol that the BSL is exposed to in 24 hour period can be determined by a percent dilution of the solvent. For example, we are using 70% methanol (ME) as our solvent. We know that we prepared the raw plant material as a 10gram/200 ml concentration.
Therefore, at an undiluted test condition of 70% ME, we would be exposing the BSL to 1g/20ml, which is 50 ug/ml.
When we perform a 1/10 dilution of 70% ME, we then expose the BSL to 7% ME and 5ug/ml of plant material.
When we perform a 1/100 dilution of 70% ME, we then expose the BSL to .7% ME and .5ug/ml of plant material.
So, first decide what treatments (plant type and X ug/ml conc.) you want to test. Then make appropriate dilutions.