Brine Shrimp Lethality Bioassay

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Procedure for cytotoxicity  test

 

Brine Shrimp Lethality Bioassay

Introduction

Brine shrimp lethality bioassay is the recent development in the bioassay for the bioactive compounds. Natural products (extracts & pure compound) can be tested for their bioactivity by this method Here the simple zoological organism (Brine shrimp nauplii) is used as a convenient monitor for screening & fractionation in the discovery of new biotic natural products. This bioassay indicates toxicity as well as a wide range of pharmacological activities of the compounds. The brine shrimp lethality bioassay has several advantages such as-

  1. Rapid in process (24 hours)
  2. Inexpensive & simple (e.g. no aseptic technique is required)
  3. It easily utilizes a large number of organisms for statistical validation & requires no special equipment 7 relatively small amount of sample is sufficient.
  4. It does not require animal serum as it is needed for determination of cytotoxicity.

Principle

Brine shrimp eggs are hatched in stimulated sea water to get nauplii. Test samples are prepared by dissolving in DMSO, desired concentration of test sample is prepared. The nauplii are counted by visible inspection are taken in vials containing 5 ml of stimulated sea water. Then the sample of different concentration are added to he remarked vials through micropipette. The vials are left for 24 hours and then the nauplii are counted again to find out the cytotoxicity of test samples.


Materials

ü  Artemia salina leaches (Brine shrimp eggs)
ü  Sea salt (Nacl)
ü  Small tank with electric air bubbler
ü  Lamp to attract shrimp.
ü  Pipettes
ü  Micropipette
ü  Glass vials
ü  Magnifying glass
ü  Test Samples of experimental plant.


Preparation of stimulated sea water (Brine water)

Since the lethality test involves the culture of brine shrimp nauplii, that is the nauplii should be grown in sea water. Sea water contains 3.8% of sodium chloride & hence 3.8% salt solution should be needed for this purpose. Accordingly 3.8% of sodium chloride solution was made by dissolving sodium chloride (38 gm) in distilled water (1000 ml) & was filtered.

Hatching of Shrimps
Sea water was kept in a small tank & shrimps eggs were taken into the divided tank, constant oxygen supply was carried out & constant temperature (37ºC) was maintained.
Two days were allowed for the shrimp to hatch and mature as nauplii. These nauplii were taken for bioassay.

Preparation of Sample
The methanolic extracts were taken for the operation. 3 mg of each extract was dissolved in 3 ml of dimethyl sulfoxide (DMSO) to get the concentration of 1µg/1µl. From this solution 12.5µg/12.5µl, 25µg/25µl, 50µg/50µl, 75µg/75µl, 100µg/100µl, 200µg/200µl, 300µg/300µl, 400µg/400µl, 500µg/500µl, were taken in a test tubes, containing 5ml of sea water &10 shrimp nauplii. So final concentration of extracts  in the test tubes were 150µg/ml, 250µg/ml, 550µg/ml, 750µg/ml, 950µg/ml, 1150µg/ml respectively.

Application of the sample & Brine Shrimp nauplii to the vials
Well cleaned vials were taken for five different concentrations. Sea water containing a number of brine shrimp nauplii was taken in each of the vials. With the help of micropipette specific volumes of the sample were transferred the vials. For each of vials contains these volumes of samples & 5 ml of brine solution contain10 nauplii.

Counting of nauplii
After 24 hours, the vials were observed, the number of survived nauplii in each vial was counted & the result was noted. For this data, the percentage of lethality of the nauplii was calculated at each concentration.

If you have any  opinion Please, comments ...

Data accumulated & compiled by:
Dewan Pavel (M. PHARM)
       Mundipharma (Bangladesh) Pvt. Ltd.
       Officer, Quality Assura




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