How to create a yeast extract from bread making yeast for medium?

[Aubrey]

Hello, I'm a high school student and I'm working on a school project to produce spider silk in vitro. Part of this project is to find the best media/ringer solution to keep the tissue alive the longest. The goal is to produce the silk at as low as a cost as possible, so I was thinking of cheap ways to get the nutrients it needs.  Since it's an organ culture, it won't last much longer than a couple of hours, but I need to supply it with amino acids so it can keep producing silk.  

So, I was thinking of using an UV light to kill the yeast then incubating it (at 45C?) for a time period. Is this a dumb idea? Will the UV light be effective in killing the yeast and not causing an infection? How do I make the extract suitable for tissue culture? 

[Scott]

Hi Aubrey,

Interesting silk project. For your question have a look at this abstract.

https://www.ncbi.nlm.nih.gov/pubmed/20819369

When making media we normally autoclave (~120oC@~20minutes) the yeast extract (and the other components). Try boiling your media for awhile if you don't have a pressure cooker.

Let us know how your project goes.

Cheers,

Scott

http://www.opensciencenet.org

[Mega [Andreas Stuermer]]

As far as I know, yeast extract is made by cooking the yeast at 55 degrees Celsius roughly. At this temperature its own enzymes such as protease and lipases degrade the cells (Autolysis). Probably afterwards it is cooked 65 degrees to kill off any non-spore forming bacteria, or autoclaved

[Abizar Lakdawalla]

hi Aubrey, what kind of cells will you be growing? Insect cells?

On Tue, Nov 1, 2016 at 6:50 PM, Aubrey <aubre...@gmail.com> wrote:

Hello, I'm a high school student and I'm working on a school project to produce spider silk in vitro. Part of this project is to find the best media/ringer solution to keep the tissue alive the longest. The goal is to produce the silk at as low as a cost as possible, so I was thinking of cheap ways to get the nutrients it needs.  Since it's an organ culture, it won't last much longer than a couple of hours, but I need to supply it with amino acids so it can keep producing silk.  

So, I was thinking of using an UV light to kill the yeast then incubating it (at 45C?) for a time period. Is this a dumb idea? Will the UV light be effective in killing the yeast and not causing an infection? How do I make the extract suitable for tissue culture? 

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[Aubrey]

I won't be growing cells really, I'll be transferring spider silk glands and spinneret to a media and try to keep them alive for as long as possible.  

[Koeng]

I've made custom yeast extract for some Mesoplamsa cultures. The instructions on how to make the media were so old that it referenced how to make your own media.

Don't do it yourself. It is a bad idea. I spent more money on purification stuff (filters and such) to get enough of it sterile than I would have paid if I got it through a company. And, may I note, this was 15% yeast extract which is 80$ for 500ml (I followed the protocol but dang). Just buy it. I'm sure there is some long lost method on how to efficiently do it out there in old literature, but if you are a high school student, just buy it.

If anyone has a better way I'm all ears, but my experience has been rather bad with diy media components

-Koeng

[Xabier Vázquez-Campos]

You can buy baker's yeast and do the extract directly from that or growing the yeast. If the later, you need to separate the cells from the culture. You shouldn't lose too much yeast filtering with some coffee filters. Afterwards, you have 2 options:
- resuspend and autoclave directly: you may want to let it settle before you use it either if you want to filter it or not to avoid all the solid cell walls before mixing with the other sterile media components.
- resuspend, cook at 45-55 °C, filter and dry the extract. Then add as you require to the medium and autoclave. You may do this and keep the suspension in the fridge for a short time but I wouldn't use it after 48h even if it was kept in the fridge

[Aubrey]

Do you know how long the extract will last with this?

[Xabier Vázquez-Campos]

Hard to say, but if you autoclave it, it should last for several weeks. As far as you keep it sterile should be good enough for your purposes. Keeping it in the fridge will help. Expect some debris on the bottom of your bottle if you store it, even if you filter it (I've seen it even in autoclaved 0.2um-filtered commercial yeast extract).

Good luck!