Media preparation

necessary tools:

necessary articles of consumption:

The sources of supply you can find under "Necessary equipment".

Culture vessels for sowing seeds (motherflasks)

We prever testtubes for seed sowing because of their length, they do do not get to hot while they are lying in the steril area (steam).

Culture vessels for replating

For replating we have to choose flasks which are about 10-15 cm high with an opening smaller than 10 cm. If the size of the opening is bigger than 10 cm the risk of contaminations increases very fast. Babyfood jars are very good.

Preparing the flasks:

Before you can use food-jars for in vitro culture you have to remove all rests of food and labels. Check if the jars can resist 150 ºC because we have to heat the flasks with this temperature for 30 minutes to kill all fungi and bacteria.

Preparing the spirit stove (only one time)

We prefer spirit stoves with solid fuel because they are easy to handle and they donīt smell bad like others do. In the picture below you can see how we have modified it.

spirit stove

Media preparation

1.

Messure out the necessary quantity of media powder.

2.

If your media does not include a gelling agent (e.g. agar-agar) you have to messure out the necessary quantity of agar-agar. For our medias we us 6 - 7 g agar-agar per liter media.

3.

Turn on the baking oven (150 ºC).

4.

Put about 2/3 of required distilled water in your beaker and add the media powder. Stir the solution till the powder is completely dissolved.

5.

Add distilled water till you reach the final quantity of media and stir well again.

6.

Measure the pH and adjust it to 5,6 (use HCL to decrease pH or NaOH to increase pH).

Sigma P6668 media is adjusted, so measuring pH is not necessary.

7.

Now you can place your beaker on the spirit stove to heat the media.

8.

As soon as the water starts boiling, stir the agar-agar powder in the water.

9.

Let the media boil for about 2 minutes and keep stirring.

10.

Dispense the media into your culture vessels. Watch out that the media does not contact the opening of the flask because this can help fungi and bacteria to contaminate this flask. A glassfunnel is very helpful.
dispense media into flasks

11.

Screw the lids loosely on the flasks. If you use test tubes you have to put a cottonplug into each test tube.
screw lids loosely

12.

Now you have to cover your flasks with aluminium foil. If you use test tube we recommend to cover all tubes with an additional aluminium foil to make shure that the cottonplug stay in the tubes. Place the vessels in the baking oven.
cover flasks with aluminium foil

13.

After 30 minutes turn off the oven but donīt open it. Let the flasks cool down in the closed baking oven. We prepare our medias always after dinner, so nobody needs the oven and the flasks can stay in it overnight.
sterilize in baking oven

14.

Take the cold flasks out of the oven and let them rest for at least 5 days to make shure that they are sterile.

Making sterile distilled water

When you want to flask dry seeds you should rinse them with sterile distilled water after sterilization. It is very easy to get sterile distilled water. Fill some distilled water in a screwable jar, screw the lid loosely on the flask and cover the flask with aluminium foil. Now you can sterilize the water together with your flasks containing the medias in the oven.

Media we use:

seed germination

P668 (Sigma P6668)

replating

P748 (Sigma P1056 without Agar-Agar)

node culture

P793 (Sigma P6793)