I am seeking advice for preparing and dispensing large batches of media for plant tissue culture. Generally I have only prepared 1L at a time in a 2L flask. However I would like to start preparing up to 10L of media at a time. I use agar, so the media must be boiled, and I also always pour my media into the vessels before running them in the autoclave. So my questions are:
What type of vessels are recommended for mixing and heating large media batches?
How do I dispense large batches of hot agar media into tissue culture vessels if the mixing vessel is too large to pick up and pour from? (I know there are some expensive media dispensers you can buy, but I am looking for lower cost options or possibly a simple design for something that I could build myself.)
Its very easy. You can use steel vessel of 5 lit capacity to boil the media. After the media is boiled add agar to the vessel and stir continuously. The agar will mix properly after 5-10 minutes. After that take 1 lit polypropylene beaker with handle and pour the molten media to beaker and then dispense the molten media from beaker to glass bottles (tissue culture bottles) and tighten the cap. Then autoclave the bottles in normal cooker or autoclave. We regularly prepare 50-60 lits of media per week by this method.
Is it necessary to heat the culture media (never boil it!!!!!!)? The heating process is time and energy consuming, in other words, heating is throw money. I prepare big batches of culture media and dispense them cold. The trick: continuos shaking and a syringe (if you don't have the possibility to have an expensive dispenser).
Thank you for the replies!
Ricardo - why do you say never boil the media? It is my understanding that agar must be boiled to go into solution. I have heard of dispensing cold and letting the agar be heated and incorporated in the autoclave to save time/energy, but I often prepare a lot of media and then run it through the autoclave at a later time, which would allow the agar to settle to the bottom of the vessels before heating. I have also been told this method can contribute to variations in agar concentration among vessels, which makes me hesitant as I have issues with hyperhydricity.
There is a difference between to heat and to boil. If you heat to much, the culture finally boil, however, it's not recomendable because some components of culture medium (carbon sources, hormones..... even agar) might change their properties. On the other hand, when I mentioned the tricks, to obtain a suitable stirring speed is very important to maintain in suspension the agar in such a way that the same quantity will be present in all the vessels. I know it's "complex" but in a couple of time you could be on the way.
I prepare 5 l at a time. Use a loarge 5 l flasks and use a hot plate. But you should not boil the media for long. Insted tou can heat it up to about 60 c. And take a portion out add your agar and mucrovave that to dissove agar. Them combine the tow. So you dont need to wait long.
If gellan gum is an option (gelrite), it is prepared at room temperature or cooler before autoclave. I used to prepare 20-40L batches using this easily.
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