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Toxicity studies in Zebrafish
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Materials Required

 

  • Sterile Petri dishes
  • Forceps
  • 24 hour embryos
  •  9-well plates
  • Screw cap tube -5ml
  • Sterile Glass pipettes
  • Micropipettes
  • Tips
  • Sterile bottles - 500 ml
  • Stereomicroscope

 

Reagents


E3 medium (50X)                 - 50ml
Distilled water                     - 1000ml
Cadmium chloride (1mg/ml)  - 5ml
Ethanol


Reagent Preparation


E3 Embryo Medium Composition (50X)


5.0 mM NaCl -14.6g
0.17mM KCl -0.65g
0.33mM CaCl2- 2.20g
0.33mM MgSO4-4.05g

 

1. Dilution of E3 Embryo Medium (50X)

  • 5 ml of E3 medium is added with 245 ml of distilled water.

 

2. Dilution of Cadmium Chloride(1mg/ml)

  • 0.5 µg/ml: 2.5 µl of cadmium chloride is added with 5 ml of diluted E3 medium.
  • 1µg/ml: 5 µl of cadmium chloride is added with 5 ml of diluted E3 medium.
  • Control: 5 ml of diluted E3 medium.


Procedure

 

  1.  A 50X stock solution of E3 embryo medium is diluted to 250 ml using distilled water.
  2.  Prepare the dilutions of cadmium chloride of 0.5 µg/ml, 1µg/ml and control for toxicity test.
  3.  Check the embryo under a stereomicroscope to ensure it is alive and looks like 24hr embryo.
  4.  The assays will be done in 9 -well plate with one healthy 24 hour embryo added to each well.
  5.  Carefully remove most of the fluid from around each embryo using micro 200 µl pipette in the LAF and replace fluid with 200 µl of control and test solution .Do carefully and quickly, do not allow embryos to dry out.
  6. The 9-well plate is kept in the incubator for 24 hours at 28.5 °C.
  7. After 24 hours, check the embryo under a stereo microscope, as the embryo is alive or dead.  If all the embryos are found dead or alive the concentration range of the test compound needs to be decreased or increased respectively.
     

 

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