Agar Anger

It has been over 7 months since I first started mushroom farming and I have yet to create a clean petri dish inoculation. So far, I have examined the following factors:

1) Set up laminar flow hood to create clean air stream

2) Used both glass petri dishes and pre-sterilized ones to ensure that petri dishes were not the cause of contamination

3) Even got pre-sterilized scalpels just in case my flame sterilization technique was not optimal

4) I sterilize the agar medium and glass petri dishes for 45 minutes and then unload it carefully in front of the flowhood (it is raised to flowhood level with a cart)

5) I am careful to wear gloves and keep everything deep inside the flowhood

6) I wipe the work area with isopropyl alcohol before I start working

7) I bought an incubator that can keep incubation temperatures at 75F for best growth

Reading through Stamet's book, Growing Gourmet and Medicinal Mushrooms, I realize that the agar plate should be colonized within a mere 7 - 14 days. It always seems like there is not much mycelial activity when I first do the inoculation - this led me to believe that it was perhaps the agar medium that I was using that was not optimal. I had been using a premix of potato dextrose agar bought from amazon, but I am planning on purchasing the individual ingredients to make the mix myself.

I will experiment with two types - potato dextrose agar and malt extract agar. I will follow Stamets' recipes exactly. In addition to the base ingredients of agar, dextrose, and malt extract/potato, he also adds in trace amounts of gypsum, peptones, yeast in different amounts for various recipes. Perhaps those are more critical than I realize for the development of the mycelium. I am hopeful that this is the reason why my growth has not been as vigorous as I see in other cultivators' labs.

There are a few other factors that could be contributing to contamination as well.

First, I have not been using a mask when working. Although I am not actively talking or singing which would expel bacteria from my mouth and possibly into the work area, it seems that wearing a mask would be prudent to make it change from minimal risk to zero risk.

Second, I have not been using a stainless steel countertop, which I know is the industry standard, but plastic sheeting which has wrinkled in various spots, which may make it easier for bacteria to remain there. If I cannot get a stainless steel sheet of some sort, I should at least get some sort of smooth vinyl or antibacterial sheeting that can be more easily and reliabily sanitized with alcohol.

Third, I may not be using the most vigorous state of growth for the mushroom for inoculation. Stamets recommends harvesting mushrooms at the button stage when they are in a "frenzied state of cell division" and then directly excising a piece of tissue from below the gill or at the base of the mushroom for most optimal growth. I thought that I was doing this correctly but wonder whether I actually am. I should watch a few more youtube videos on this to confirm proper technique.

Fourth, I also have a vertical laminar flowhood which pushes air top down. When I unload the All American sterilizer which sits on top of a cart that makes it the same level as my workspace area, there is a split moment when it is not covered by the air flow. If I push the laminar flowhood a few inches over the edge of the workspace, it would kind of cover that gap as I unload the sterilizer and make it less likely that contaminated air flow in.

I feel that once I fix the above factors, this should resolve my issues. If all of the above changes still result in no success, I will be flaggerbasted and discouraged, but hopefully it is not the case. Then I will be able to finally move on to liquid cultures and grain spawn!