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Results

Effect of Hemin (Iron) on the growth of Leishmania

Iron is a necessary component for the growth of Leishmania. Hemin is one of the important sources of iron (Fe⁺³) for the invitro culture of Leishmania. The aim of this experiment was to test the effect on the growth of Leishmania if we decrease the amount of hemin (from its optimal) in the Leishmania culture media.

1. We made the Leishmania culture media (M199) without adding Hemin.

2. We made Hemin stock solution. Hemin is not soluble in water so we dissolve it in 1.4M NaOH solution to make the stock solution.
       Solubility of Hemin in 1.4M NaOH is 25 mg/mL.
       Molecular Wight of Hemin is 651.94 g/mol.
       We dissolved 25 mg of Hemin in 2 mL of 1.4M NaOH solution to make the stock of Hemin. So, the final concentration of Hemin stock solution is 0.02M.
   

3. Then we aliquoted the previously made M199 media in ten different falcon tubes and added different volume of Hemin (100% or the optimal concentration, 75% of optimal concentration, 50% of optimal concentration, 25% of optimal concentration, 0% or no Hemin) from the stock solution (two falcon tubes for each Hemin concentration).

4. Then we cultured Leishmania major in these media of different Hemin concentrations.

5. We counted the number of cells (in Hemocytometer) in every 24 hours for 3 days.

We started with the same number of cells in each culture media.

24 hours later...

42 hours later...

72 hours later...

So, we can conclude due to the absence of Hemin the growth of Leishmania is affecting. Probably this growth is due to the iron which is present in FBS (used to prepare Leishmania culture media).