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OfflineInsultingLizard
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Registered: 01/04/20
Posts: 546
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Question about sterilization methods
    #26469797 - 02/05/20 07:43 AM (4 years, 11 days ago)

After having some issues with contamination in my current batch of PF jars, I want to improve my sterilization procedure. I think my problem was either that I steam-sterilized the shot glasses for the spore prints rather than pressure-cooking them, or that the syringe with the boiled water got dirty.
Unfortunately, the largest PC I can buy is only 11 liters and only goes up to ~11.5 PSI. To get a big sterilization chamber that goes to 15 PSI I'd have to go all out and buy an autoclave, the cheapest of which is almost four times as expensive as a PC.

So my options are:
* Keep my pot and do tyndallization (aka fractional sterilization).
* Keep my pot and do steam-sterilization over 8 hours. Some members have suggested this is less time-consuming than sterilization and at least as effective.
* Buy a small PC and adjust sterilization time to account for the lower pressure. Also sterilizations will need to be done in smaller batches.
* Bite the bullet and buy an autoclave.

What do you guys think? I may buy an autoclave eventually if I do lots of grain sterilizations, but at this time I think it's too large an investment for me.


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Offlinemigraineur
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Registered: 02/15/10
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Re: Question about sterilization methods [Re: InsultingLizard]
    #26469808 - 02/05/20 07:51 AM (4 years, 11 days ago)

Why can't you buy a Presto pressure cooker or All American pressure cooker? They will go to 15PSI and can be bought in large sizes. They're cheaper than fully fledged autoclaves.


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OfflineInsultingLizard
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Re: Question about sterilization methods [Re: migraineur]
    #26469829 - 02/05/20 08:09 AM (4 years, 11 days ago)

I don't live in the US and currently imports are restricted, and even if they weren't the shipping costs would be prohibitive.


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Invisiblepppp
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Registered: 10/11/16
Posts: 45
Re: Question about sterilization methods [Re: InsultingLizard]
    #26469865 - 02/05/20 08:31 AM (4 years, 11 days ago)

Hi InsultingLizard,

What were you using until now to serialize?

I can't tell you what to buy, but I can share my experience so far.
I started off with what I had, which was a very old, steam leaking, would only fit one-quart jar at the time pressure cooker.
I don't even know what kind of pressure it reaches + it's leaking a lot of steam during the process.
Never got contaminated grain.

In your place, I would look around in second-hand markets or garage sales or get one from friends, co-workers or family, just make sure whatever size jar you want to sterilize fits in it.
Just my opinion.
I hope it helps.


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OfflineNeowynd8
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Registered: 06/12/16
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Last seen: 7 months, 19 days
Re: Question about sterilization methods [Re: InsultingLizard]
    #26469873 - 02/05/20 08:35 AM (4 years, 11 days ago)

Lack of pressure cooker isn't your problem. You don't even need one for PF Tek. Steam sterilizing is fine. You're problem is your inoculant or technique.


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Invisibletramalot
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Re: Question about sterilization methods [Re: Neowynd8]
    #26469917 - 02/05/20 09:05 AM (4 years, 11 days ago)

Spores are "dirty" by nature, try Agar maybe. I know if your substrate is to wet or on the wet side contaminants can grow quickly aggravated by the extra water in the syringe.


--------------------
It's the Journey I enjoy!


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OfflineInsultingLizard
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Registered: 01/04/20
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Re: Question about sterilization methods [Re: InsultingLizard]
    #26469959 - 02/05/20 09:39 AM (4 years, 11 days ago)

Quote:

You're problem is your inoculant or technique.


That's my guess, but I'm unsure what it might have been.

* I wrapped the shot glasses in aluminum foil and sterilized them with the jars.
* I boiled tap water in a pot for 10 minutes and sucked it into a syringe, left it for two minutes, emptied it, repeated 3 times and then stored the syringe in the fridge until next day (when I did the inoculation).
* I did all my work in a SAB with gloves and Tyvek sleeves. I disinfected the box with antibacterial cleaner before starting and sprayed the gloves and sleeves with alcohol every time I took them out of the box.
* I used new sterile syringes for the spores and I sterilized with an alcohol torch the needles and the inoculation loop.

Still, 4 out of 7 jars didn't colonize at all, and instead developed this moist, dark look in the substrate directly below the inoculation holes.


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OfflineInsultingLizard
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Registered: 01/04/20
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Re: Question about sterilization methods [Re: InsultingLizard]
    #26470035 - 02/05/20 10:33 AM (4 years, 11 days ago)

By the way, how do you inoculate cakes with agar? I understand that you open the jar and drop the slice of agar in, but what do you do about the vermiculite filter? Do you not add it, do you add it after the agar, or what?


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Invisibletramalot
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Re: Question about sterilization methods [Re: InsultingLizard]
    #26470056 - 02/05/20 10:44 AM (4 years, 11 days ago)

Go down to a Chinese restaurant if they have any and see what they use for rice... Cheap pressure cooker's should be everywhere...


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It's the Journey I enjoy!


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OfflineNichrome
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Registered: 12/17/18
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Re: Question about sterilization methods [Re: tramalot]
    #26470101 - 02/05/20 11:08 AM (4 years, 11 days ago)

11 psi just needs a slightly longer cook time to achieve the same results. The problem is what you did with your syringe. If your boiling water to use in a solution you want to put it in a clean syringe right before you use it.

Get a mason jar, poke a small hole in the lid and cover with masking tape, pour boiling water in the jar, then let cool. It will be a vacuum sealed jar of sterile water when it cools. You can peel up the tape and fill your sterile syringe when cool (in an SAB or in front of a hepa filter). Steam sterilization works fine for the syringe. Wrap the syringe in foil and steam it. Only fill it up when you're ready to make solution. Use solution right away.

without proper gear everything is still doable, you are just racing contams a little more. Anything you can do to speed up the time from inoculation to fruiting the more likely you'll achieve better results.

This should help quite a bit with limited tools.


--------------------
Better to be deprived of food for three days, than tea for one.


Freedom is not the right to do as you please, but the liberty to do as you should. ~Emerson


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OfflineInsultingLizard
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Registered: 01/04/20
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Re: Question about sterilization methods [Re: Nichrome]
    #26470137 - 02/05/20 11:34 AM (4 years, 11 days ago)

Quote:

The problem is what you did with your syringe.


Yeah, I figured that was the most likely culprit.
Thanks for the tips, Nichrome. I'll follow your advice next time I prepare syringes.
I guess I'll hold off on the PC until I'm doing grains.


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