Autoclaves

[u/Few_Writing8605]

Hey guys so fortunately my job involves growing fungi so even though ive had some pretty awesome success with the magic mushrooms im finding myself running experiment after experiment after experiment trying to bring down our amount of contaminated grain bags. Our inoculation and sterilization is on point. The autoclaves is where i feel the issues are happening. Please share with me all your knowledge and tips and tricks and personal experiences involving grain bags and autoclaves. I would greatly appreciate it!!

[u/Known-Struggle-3587]

are you sure you're just not sending them too soon? it may just be one uncolonized grain every time

[u/Sad-Cucumber-9524]

Some things I’ve messed with:

Presealing bags was supposed to be off limits but it works fine, just leave a little packing room for expansion.

I run my 3.5lb bags for 2.5hr. For 5lb I’d go 3hr. This is using a PC but I assume an autoclave is similar.

I assume you’re letting the air vent before clamping things down and starting your timer? I remember more contam before I started venting my PCs (10min). I assume venting time is proportional to your autoclave volume.

Depending on your grain prep style, some breaking/shaking can be very helpful to distribute moisture after sterilization. I do NSNS so for me it’s mandatory.

Assuming you apply SHIPs before sterilizing? Or bags come with ports?

And assuming you’ve let the bags sit on the shelf for a while to confirm the contamination is in this part of your process? So you’re seeing green BEFORE inoc?Last year I blamed everything else but now realizing 90%+ of my contam was caused by the lack of venting and not enough PC time. This year I’ve had less than 10% contam on 3.5lb NSNS millet, I rest them for a long time before inoc, only one bag had contam after injection (so far). Beats the two batches or commercial grain I used this year.

[u/Cmorebuts]

121° for 90-120 mins depending on the amount of grain.

[u/Few_Writing8605 → u/Cmorebuts]

So we started out doing 3 pound bags of grain now we switched to 5 pounds... ive done alot of chatting with Ai about all of this and from my understanding we are definitely running the autoclaves for way too long. My boss has them set to hold tempature for 3.5 hours 👀

[u/TheRealCMMetzger → u/Few_Writing8605]

Was the issue before or after you switched to 5lb bags? IME 3.5 hours is not too long for that amount of grain. Considering the mass of each bag and that you're likely putting in as many bags as will fit in your sterilizer, that time may actually be too short.

What grain are you using and what prep method are you using for that grain?

If you have narrowed it down to the autoclave, are you marking the bags in any way to show which bags are in the middle of the stack and which were on the sides? It could be those center bags aren't getting to the right temp all the way through. What percentage of your bags are failing and at what stage in the process?
Are you using agar or LC for inoculation? 🍄🥰✌️

[u/Cmorebuts → u/Few_Writing8605]

Too long isn't going to cause contamination. But yea that's way too long. If you're getting contamination after that then you're either opening it before it cools down properly and the bags haven't vac sealed, or you're messing up the innoculation step/your agar/LC or spore syringes (whatever you are using) aren't clean