Agar Anger - Part 2

Another attempt at pouring agar has yielded improvement but other problems. One difficulty with pouring agar is the trick of pouring it right before it congeals into a solid state. I was using a no-contact digital thermometer to read temps as I read optimal pouring was between 120 - and 130F, but it seemed to not read the fluid temperature in the glass jar very well. By pouring between those two temperatures, you avoid condensation on the agar plates, which can sometimes be an impediment to mycelial growth. With the most recent batch, I waited too long, and about half of the bottle turned into jelly before I was able to pour it. Next time, I plan on using a timer for all steps, mainly to record two things: (1) length of sterilizer cooling post sterilization and (2) length of agar cooling in front of flow hood directly before pouring. I can then decrease the cooling time if it gels again.

Prior to this agar pouring, I had implemented a few changes, which I think should significantly boost my success rate. They are listed as follows:

• I created a separate lab room in my house, so I am not mixing the fruiting area (dirty and lots of in and out movement) and lab area;

• I am starting flow hood at least hour prior to pouring agar to reduce number of contaminants in the air (tip I read somewhere)

• I am making sure to use a mask when pouring - yes, I was aware of this always but was just lazy and sometimes did not wear

• I am sterilizing agar with magnetic piece inside and then use stir plate to mix liquid for evenness prior to pouring

• I am making sure to wipe down surfaces of everything with isopropyl alcohol before beginning my work. This includes plastic sleeve holding the sterile petri dishes, stainless steel surface outside of sterilizer

All in all, I just get the general sense that my new set up is good enough for me to be able to get to a 90%+ success rate. It is likely that the last vector of contamination will not be lack of equipment but rather lack of aseptic technique from the cultivator (aka myself).

In my understanding, there are three main "ideas" when it comes to working in front of a flow hood to minimize chance of contamination. First, more sterile substances should always be placed closest to the flow hood. For example, if a less than sterile item - even it has been sanitized with isopropyl alcohol - is placed behind the sterilized agar plate, there is a chance that contaminants from the only sanitized item float over the agar plate and nestle into it. So cleaner => in back; dirtier => in front. There may be cases where there are exceptions to this rule, but as a matter of general principle, it holds true. This also includes hand movement, whereby the cultivator's hands should always be in front of and to the side of sterile cultures.

The second idea is working within the confines of the flow hood and minimizing hand movement. Every time one's hands leave the flow hood area, there is a chance that contaminants settle on them. I should be careful to minimize the number of times my hands need to leave the center of the flow hood area so as to ensure exposure to contaminants. Additionally, slow and deliberate hand movements minimize air flow around them, reducing the chance of picking up stray unwanted particles in the air. Naturally, this will come with time and experience.

Third, every time, something does leave the flow hood area or is otherwise made unsterile by contact with something else, it should be immediately re-sanitized/re-sterilized. For example, if the cultivator needs to grab something outside the laminar flow hood wind stream, they should re-sanitize their gloved hands with isopropyl alcohol immediately before continuing work. Or, if a scalpel is used to press mushroom tissue into an agar plate, it should re-flame sterilized before further use. It follows that items like phones which are a significant vector of contamination should usually be left outside the lab while working.

Even all the above probably seems trivially basic to the experienced wet-lab worker. I realize that I am very far from producing absolutely amazing 100% contaminant free cultures. But there is hope! I am particularly interested in continuing to learn from Gary from Fresh From the Farm Fungi who has a wet-lab background and almost exclusively sells various mushroom cultures (agar, liquid culture, etc.) online. I did buy his book where he focuses primarily on the lab work, mainly the preparation of agar, liquid cultures, and grain spawn, the three big lab items for mushroom cultivators.

With the mix of learning from more experienced mushrooms cultivators, watching YouTube videos, reading, and hands on application, my lab skills will surely get better.