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0.5x MS Media with MES Buffer Protocol

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0.5x MS Media with MES Buffer Protocol

1. Purpose:

This protocol outlines the preparation of 0.5x Murashige and Skoog (MS) salts media supplemented with MES buffer, which is suitable for plant tissue culture applications.

2. Materials:

* Chemicals:

  • MS Salts powder (includes macro and micronutrients)
  • MES hydrate powder (2-(N-morpholino) ethane sulfonic acid)
  • Potassium hydroxide solution (0.5 M KOH)
  • Plant Agar powder

* Equipment:

  • Analytical balance
  • Magnetic stirrer and stirring bar
  • Graduated cylinder (3 L)
  • Volumetric flasks (2.5 L, 500 ml x multiple)
  • pH meter with calibration solutions
  • Beaker
  • Pipette
  • Autoclave

* Personal Protective Equipment (PPE):

  • Lab coat
  • Gloves
  • Safety glasses

3. Procedure:

1. Preparation:

Thoroughly clean and dry all glassware before beginning the protocol.

2. Dissolving MS Salts & MES:

  1. Using the analytical balance, weigh 6.45 g of MS salts powder and 1.5 g of MES hydrate powder.

  2. Transfer these weighed powders to a clean beaker containing approximately 2 L of distilled water.

  3. Place the beaker on a magnetic stirrer and stir continuously until both powders are completely dissolved.

3. pH Adjustment:

  1. Carefully calibrate the pH meter using standard calibration solutions following the manufacturer’s instructions.

  2. Slowly add 0.5 M KOH solution to the MS salts/MES mixture while stirring continuously. Monitor the pH with the calibrated pH meter.

  3. Adjust the pH to a range of 5.6-5.8. Record the final volume after pH adjustment for accurate results.

4. Final Volume Adjustment:

  1. Carefully transfer the adjusted solution to a clean 3 L volumetric flask.

  2. Add distilled water to the flask until the meniscus reaches 3 L. Mix thoroughly to ensure homogeneity.

5. Aliquoting and Agar Addition:

  1. Aliquot the prepared MS media into multiple sterile 500 ml bottles.

  2. To each bottle, add 4 g of plant agar powder. This will ensure a final agar concentration of 0.8% after autoclaving.

6. Sterilization:

  1. Autoclave all bottles containing the MS media and agar at 121°C for 15 minutes to sterilize the solution.

7. Agar Mixing:

  1. After autoclaving, carefully invert each bottle several times to ensure the agar is evenly distributed within the solution while it’s still hot.

8. Storage & Use:

  1. Allow the sterilized bottles to cool down to room temperature (RT).

  2. Store the bottles at RT until needed.

  3. Before use, microwave each bottle for a short period, carefully monitoring it, to melt the agar and create a homogeneous solution.

End of Protocol.

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