Plate to...?
Tissue cloning - a learning experience
My cloning practices are primitive, but so far surprisingly effective. I added tissue to the plates immediately after boiling the agar, so as to minimize the number of times I opened the plates and risked contamination. I've made a small start with 8 plates of oyster mushroom from Whole Foods, 8 of Agaricus bisporus, and 8 control (no mushroom added). I mixed my agar with varying amounts of H2O2, from none to, er, quite a lot.
Results:
All my controls stayed clean. Yay!
The crimini (AB) also stayed clean. it won't grow in the presence of more than minimal H2O2. I have 3 samples that show substantial growth.
The oyster mushroom itself was contaminated. A small "pool" of bacteria grew over the flesh of each OM sample. However, in two cases, the bacteria did not spread any further; there is a nice flat ring of OM mycelium reaching out to the edge of the plate.
Two samples that I foolishly opened for photography were severely contaminated and lost.
So my process is good (surprisingly good for not having a glove box or autoclave), except for the photography attempt, but my oyster mushroom tissue sample was not so good.
I want to proceed with my experiments using the two minimally contaminated OM cultures. I have an experimental technique for sterilizing grain and sawdust without the use of an autoclave; it's the same technique I use to sterilize soil for starting plants in mini-greenhouses in order to avoid damping off, and it works quite well for that (except that the plants have difficulty adapting to the outside world after having grown up in such a sheltered environment).
I'll place grain or sawdust in a heatproof, non-metal container with the appropriate amount of water, then seal the container. It will be microwaved - I'm not sure how long for; I usually went for two 2:00 runs when sterilizing soil. I'd also like to try mixing my grain with 151 and letting it sit before adding water and microwaving.
I think I'll do this with some controls and let them sit for a while. That will tell me if my technique is sound before I risk my cultures.
I wonder if I need to re-culture on agar first, or can I go straight to grain spawn with the clean-looking part of the plates. Also, is it actually sensible to *do* the grain spawn step with oysters, or would I be better off going straight to sawdust?
I could prepare my grain spawn and my agar and do one big culturing day! That will be a lot of work, though.
My cloning practices are primitive, but so far surprisingly effective. I added tissue to the plates immediately after boiling the agar, so as to minimize the number of times I opened the plates and risked contamination. I've made a small start with 8 plates of oyster mushroom from Whole Foods, 8 of Agaricus bisporus, and 8 control (no mushroom added). I mixed my agar with varying amounts of H2O2, from none to, er, quite a lot.
Results:
So my process is good (surprisingly good for not having a glove box or autoclave), except for the photography attempt, but my oyster mushroom tissue sample was not so good.
I want to proceed with my experiments using the two minimally contaminated OM cultures. I have an experimental technique for sterilizing grain and sawdust without the use of an autoclave; it's the same technique I use to sterilize soil for starting plants in mini-greenhouses in order to avoid damping off, and it works quite well for that (except that the plants have difficulty adapting to the outside world after having grown up in such a sheltered environment).
I'll place grain or sawdust in a heatproof, non-metal container with the appropriate amount of water, then seal the container. It will be microwaved - I'm not sure how long for; I usually went for two 2:00 runs when sterilizing soil. I'd also like to try mixing my grain with 151 and letting it sit before adding water and microwaving.
I think I'll do this with some controls and let them sit for a while. That will tell me if my technique is sound before I risk my cultures.
I wonder if I need to re-culture on agar first, or can I go straight to grain spawn with the clean-looking part of the plates. Also, is it actually sensible to *do* the grain spawn step with oysters, or would I be better off going straight to sawdust?
I could prepare my grain spawn and my agar and do one big culturing day! That will be a lot of work, though.