In making a tissue culture medium, you are required to use final concentrations

Question

In making a tissue culture medium, you are required to use final concentrations of 10M indole-3-butyric acid(IBA) and 3M 6-benzylaminopurine(BAP) in 1L of final medium. The formula weight of IBA is 203.2 and the formula weight of BAP(as hydrochloride) is 261.7. Write out a protocol to explain exactly how you would achieve this, including what masses(weights) of solids you would add to what volumes of which liquids and any stock solutions and dilutions you would have to make. At what stage in your medium preperation would you add the growth regulators?

Explanation / Answer

Preperation ans sterilization of a culture medium:

Dissolve MS mixture in 800ml of water, stir the water continuously while adding the salt mixture.Add 30 g of sugar and stir. Adjust pH to 5.8 using 1M NaOH or 1M Nacl. Add distilled water to make the volume upto 1 litre. Weigh out 8 gms of agar and add it to the MS solution. heat it gently , pour the warm medium intopolycarbonate tubes. Place the tubes in a pressure cooker and and sterilize for 20 mins. When using IBA , the um for 1mg/ml is 4.90 and forBAP is 3.82. FOr BAP the solvent will be water, and for IBA the solvent will be EtOH/1N NaOH . For 10 um IBA it should be 49 and for 3 um BAP it should be 10.86.

Stock solution preperation

To prepare 1 mh/ml stock solution add 100 mg of growth regulator to 100 mg of volumetric flask , add 2-5 ml of solvent to dissolve . After it is completely dissloved bring to volume with double processed water. Add 1ml of stock solution to 1l of medium to obtain final concentration.

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