User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/09

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GARDENING/Preparation of flasks for inoculated seeds


Preparation of Fahraeus solution with agar to put inoculated seeds.

  • Gustavo and I continue with gardening activities. Today we prepare flasks where we are going to put the germinated seeds after inoculate them with 4 different strains of Rhizobium etli, and also seeds not inoculated that will serve as control. The flasks must contain a solid medium prepared with agar and Fahraeus solution, this solution contains elements that will help the plant to grow and develop well.

The first thing we do was to put 1.65 g of bacteriologic agar in 30 flasks of 250 ml. We use 30 flasks because we are going to inoculate 6 plants with every strain and we are going to left 6 plants uninoculated. The next step was to prepare Fahraeus solution, we have to prepare 6600 ml of solution because each flask should have 220 ml. The components of the solution are prepared in stocks, so then you take a volume of the stocks to prepare 1 L, 2 L, etc. The general proportions to prepare the stocks, and the volume that should be added to a L of solution are the following:

Component Stock (g/L) Volume for 1 L
MgSO4·7H2O 12 10 ml
Na2HPO4·12H2O 15 10 ml
KH2PO4 10 10 ml
CaCl2 10 10 ml
Ferric citrate 0.5 10 ml
Traces 1 l

To prepare the 6600 ml of Fahraeus solution we first put 2200 ml of water in 3 plastic flasks of 4000 ml. Then we added to each flask 22 ml of each stock and 2.2 ml of traces. After this process we fill the 30 flasks to which we had previously thrown agar with 220 ml of the Fahraeus solution. Then we put the 30 flasks in the autoclave and once they were out we left them in Esperanza´s laboratory all night.