Agar Technique

One of my initial goals was to do my own grain spawn in house - alas six months has passed and I have yet to get there. The bottle neck is the agar inoculations with mushroom tissue. If I can someone get passed this and produce reasonably good agar plates with minimal or no contamination, I believe the grain inoculations would fall inline.

I continue to make changes to reduce the chance of contamination. Most recently, I have made these changes:

1. Getting a cart that I can place the sterilizer on top of, so that I can open the sterilizer right in front of the laminar flow hood

2. Flame sterilizing blade for a longer period of time;

3. Sealing glass petri dishes with parafilm after inoculation to prevent contamination, then storing them upside down to reduce condensation on agar media, which can facilitate bacterial infection

4. Buying an incubator to keep plates at around ~75F, which is ideal for oyster/lions mane mycelium, which I'm cultivating.

Unfortunately, these changes were not nearly enough and my most recent batch of ~20 petri dishes were ALL contaminated. This points to obvious major mistakes on my end, due to my lack of chemistry/bio/lab background.

Given that most if not all petri dishes were contaminated, I believe the most likely culprit is scalpel. I had been using the scalpel for multiple purposes and exposing it to all variety of things. Although I wiped it with alcohol and flame sterilized it before use, it is likely that traces of bacteria and other small particulates remained (perhaps due to improper sterilization technique on my end). Regardless, I was advised to maintain a separate scalpel just for inoculations and I also bought a batch of pre-sterilized disposal scalpels. Additionally, I was also advised to soak the scalpel for up to 24 hours before use, even before flame sterilization. So for my next batch, I will use both the soaking method for main separate scalpel and the pre-sterilized disposable one. This should greatly reduce contamination if indeed scalpel is the cause.

Secondarily, it is possible that my glass petri dishes were not completely sterilized after 45 minutes of sterilization at 15PSI. This seems less likely, but just in case, I also bought a pack of pre-sterilized plastic petri dishes. I will do one batch with these using the new scalpel method and one batch with the glass using the new scalpel method. This should provide me with information on whether the glass petri dishes were the culprit.

Third, it is possible that (1) the laminar flowhood is not functioning completely properly and is not actually providing clean air or (2) the surface of the work area is not completely clean. Therefore, I should wipe down the surfaces with isopropyl alcohol more thoroughly before inoculation. These factors seems less likely, however, given that most of my dishes were contaminated. I would be surprised if the relatively new flowhood was not functioning.

Still, a couple related changes I could make is working more deep into the flow hood and and then also reducing the size of the front opening so that contaminants cannot somehow float back in, but I suspect that the scalpel or petri dishes are the more likely culprits to be honest.

Finally, one additional change I should make is testing blanks - if I do not inoculate the agar medium with mushroom tissue and there is no contamination, that means that the scalpel and inoculation process is the problem. This is very valuable information as it would mean that my sterilization process and agar pouring is alright and the flowhood/surfaces are not the main problem.

Although I have been quite frustrated by my lack of success in this area, I feel that I know have a much more nuanced understanding of agar inoculations and have a feeling I will create my first successful plates very soon.

Few other tips:

• Flame sterilization should really only take 3 - 5 seconds based on my research

• Again, make sure to have dedicated scalpel for inoculations and to soak overnight before usage

• Select tissue from inside of base of mushroom or right above gills for most vigorous growth per paul stamets - a tiny grain sized piece is enough to grow out.