sterile plants in tubes

Preparation of plant culture media

General Instructions

1. Measure out approximately 80% of the final volume of deionized water, e.g. 800 mL for a final volume of 1 liter.

2. Weigh in all heat-stable ingredients according to the culture media formulation. In a sequence add nutrients, iron-EDTA, vitamins, myo-inositol, growth regulators (if thermostable and autoclavable), organic supplements, sucrose. Stir the solution until completely dissolved. Heating may be required to bring all components into solution.

3. Adjust the pH value of the medium to the required value (e.g. pH 5.8 for MS) by adding dropwise solutions of 1N KOH, NaOH or HCl while stirring.

4. If required add agar (or other gelling medium) to the medium while stirring. If a gelling agent is used, heat until the solution is clear.

5. Stir the solution until the solution is clear. Add deionized water to the final volume.

6. The medium is dispensed in glass or polypropylene vessels and plugged with cotton plugs and metal caps.

7. Sterilize the culture medium in an autoclave for 20 min at 121° C at 15 psi (105 kPa).

8.  Allow medium to cool prior to use.

If the medium contains heat-labile substances:

Follow step 1 – 5 except for the addition of heat-labile substances.
Prepare culture medium with only the heat-stable ingredients and sterilized as such in a big flask or bottle.

Filter sterilize solutions of the heat labile compounds (e.g. certain vitamins or plant hormones) using a 0.22 μm filter unit. After autoclaving, the medium is kept in a laminar flow hood and allowed to cool to approx. 50° C. The required quantity of the sterile compound is added to the medium with sterile
micropipettes. Mix well the completed medium. The medium is then dispensed into sterile containers (generally sterile petri dishes) under the hood, provided the neck of the Erlenmeyer flask is passed over a flame before the medium is poured from it. Medium is allowed to cool and solidify in a laminar airflow hood.

Storage of Media:

After cooling, store media containers at 4-10° C (recommended) or at room temperature. Observe the media for 3 – 4 days after preparation, so that contamination will start to appear if the media are not properly sterilized.

Notes

Prepare powder mix of plant media only in quantities that will be consumed in a short time. Use the entire batch to prepare a solution. The removal of partial quantities is generally not recommended, as the bulk density of the ingredients can be very different, resulting in the rapid separation of components of a powder medium. In addition, powder media are extremely hygroscopic. Accurate quantities are also important because plant culture media often contain high concentrations of mineral salts which tend to precipitate in solutions.A small amount of precipitate is normal, it is mainly due to phosphates, iron or zinc complexes and does not affect the function of the media.


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