Lethality Bioassay Using Artemia salina L.

This protocol is a very useful tool for a preliminary general toxicity evaluation on different types of samples, such as extracts, fractions, and compounds from different natural or synthetic sources. The main advantage of our protocol is that it’s very easy and cheap and can be used on many samples at the same time. Moreover, the handmade equipment improves the reliability of the site.

In this technique stage, we do a screening to select the samples based on their preliminary general toxicity profile that will undergo further specific tests, such as expansive cell lines or even in-vivo tests. This method is mainly exploited in medicinal and other products chemistry fields to screen biotic samples. Our protocol can also be used for other kinds of samples such as nanosystems.

While performing for the first time, be patient because this needs fine observation skills and a lot of attention to all the parameters reported Marcia Filipe and Vera Isca, the PhD students from our lab will help to demonstrate this procedure. Begin by placing the funnel shaped container in the black support and turning the funnel in a suitable direction to see the level mark and the tap. To make the handmade migration equipment cut the top of two 0.5 liter plastic bottles to obtain a final height of 12 centimeters.

Create a hole of 1.5 centimeter diameter on one side and seven centimeters from the bottom of each bottle and insert a 13 centimeter rubber tube between the two openings. Seal the openings with hot glue and leave them to dry for 15 minutes. Put the bottles on a flat surface and fill them with water to verify there is no leakage.

Add 28 grams of the salt to 800 milliliters of tap water to prepare the artificial salt solution, or brine shrimp salt at a concentration of 35 grams per liter. Mix it with a stirring rod until all the salt is thoroughly dissolved. To prepare the samples dissolve a suitable amount of each extract and compound dimethylsulfoxide, or DMSO, at a final concentration of 10 milligrams per milliliter.

Then dilute 10 microliters of each sample in a new micro centrifuge tube using 990 microliters of the artificial saline solution to obtain a final concentration of 0.1 milligrams per milliliter. Under a fume hood in an Erlenmeyer flask, prepare a solution of potassium dichromate in distilled water at a concentration of one milligram per milliliter. Fill the hatching vessel with the medium up to the 500 milliliter mark and add one spoon, or approximately 0.5 grams of brine shrimp cysts to the salt solution.

Close the container. Place a lamp pointing directly toward the equipment and turn it on. Attach the air supplier system to the connector placed on top of the equipment and turn the pump on.

Brine shrimp cysts hatch in the artificial salt solution under vigorous aeration, continuous lighting, and stable temperature. After 24 to 48 hours, when the eggs have hatched, turn the air pump off and wait until the nauplii are separated from the empty egg cases. To separate the unhatched eggs from the alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the handmade migration equipment container.

Ensure that the solution containing the nauplii and the residual unhatched eggs is below the tube level. Place the equipment in the incubator for four hours, then in the second container add the residual salt solution above the height of the tube. Cover the container with nauplii and the residual unhatched eggs using aluminum foil and place the lamp on the second container with just the salt solution.

The brine shrimp will be attracted by the light and migrate from one container to another, leading to efficient separation between eggs and alive artemia. From the harvesting container, collect 900 microliters of saline solution containing 10 to 15 nauplii and place the solution in one well of a 24 well plate. All the samples are tested in quadruplicates.

Add 100 microliters of each negative control. Positive control, the artificial salt solution, and each of the samples to the wells and incubate the plate under illumination for 24 hours. After 24 hours, register the number of dead larvae in each well under a binocular microscope at 12 x magnification.

Add 100 microliters of potassium dichromate solution to induce the death of the remaining living larvae and wait for six hours. Count the total dead larva in each well under a microscope and determine the mortality rate according to the equation shown on the screen. Perform all the tests in triplicates and calculate the standard deviations.

Finally, express the results as the mean of three independent experiments each with internal quadruplicates plus, minus standard deviations. The structure of the selected compounds extracted from Plectranthus species and obtained by semi-synthesis are shown in this figure. A newly hatched nauplii of Artemia Salina as seen under the microscope is shown in this image.

This graphical image represents the mortality rate of Artemia Salina after 24 hours of exposure to four methanol extracts of Plectranthus species at 0.1 milligrams per milliliter. All the extracts were acceptable in terms of general toxicity using this assay. Here salt corresponds to the salt solution or blank.

Potassium dichromate was used as a positive control and DMSO was used as a negative control. All the tested extracts showed very encouraging results with very low values of mortality percentages comparable to those registered for the blank and the negative control. The mortality rate of Artemia Salina after 24 hours of exposure to the five pure compounds at 0.1 milligrams per milliliter is presented in this figure.

From this data, it is evident that only five shows minimal toxicity using this assay You need to remember three steps in particular, hatching, migration and collection. Consequently, we have other shrimp separated from and the numbers to work with. The preliminary site is a simple and low cost model channel toxicity compared to other sites like cell culture or zebra fish.

Further, more expensive cytotoxicity sites can then be exploited.