Dipping My Toes into Lab Work

Yesterday, I sterilized an agar medium and 10, 90mm glass petri dishes for the first time. I am finally getting started with trying to create my own spawn in house so as to minimize the costs of buying spawn from third-party suppliers. Honestly, I had kind of been dragging my feet on this, so I'm glad I actually went through with the motions yesterday.

And it wasn't even that hard! Except for the fact that I messed a number of things up. Reflecting on yesterday's experience, the following is a list of things that I could have done better.

1) Open up the sterilizer in front of the flow hood. I keep my Aller American 50X sterilizer on the floor, so I had to open it up near by but not directly in front of or underneath my laminar flow hood. I just purchased a cartable table with wheels. This will enable me to elevate the sterilizer to the level of the flow hood and then open it right in front of it, minimizing contamination from airborne spores

2) Once opening the sterilizers, I should let the glass jar with agar and media cool to the point that I can handle them with latex gloves. This way, I can sanitize my gloved hands with isopropyl alcohol before handling. Yesterday, I tried to remove them when still hot, so ended up using oven mitts, which are more difficult to sanitize.

3) I should pour all the plates in quick succession one at a time. Removing one of the petri dishes from the sterilizer at a time, I should pour the agar medium into each one quickly before moving onto the next. Yesterday, I was really clunky with my movements and moved them all out onto the table first before pouring them all one by one. What I'm saying is that as I move them out of the sterilizer, I should pour agar into them to reduce the steps even further.

4) I should let the agar in each of the plates cool first to the point of solidification before inoculating with mushroom tissue. Yesterday, I did not let them cool, and inoculated with tissue in the hot poured agar. This stresses the mycelium, which will damage if not outright kill it, preventing proper growth of mycelium in days following.

5) I should flame sterilize the scalpel before each inoculation. Yesterday, I only flame sterilized it once in the beginning.

6) I should seal each plate with parafilm once inoculations are done to minimize chance of any airborne contaminants entering the petri dishes.

7) I should store the petri dishes upside down to reduce condensation on the agar medium, which would increase the chances of bacterial growth.

8) I should only use the inner tissue as close to the base as possible from smaller mushrooms in the button stage, as this is when they are in their most frenzied state of cell division. Yesterday, I did not just use the inner tissue from the base but used the outside as well. I also believe I used too much when a rice-kernel sized piece is enough to start the mycelial growth

By following all of the above procedures, I will greatly increase the chance of successfully growing tissue in agar. I will be quite surprised if any of 10 petri dishes I inoculated yesterday grow out without any bacterial infection given the horrendous number of mistakes I made throughout the entire process. I am sure as I come to become an expert in agar inoculations that I will soon identify more points of improvements.