Inoculating went wrong

[Elpo]

Hi guys,

Today I made a few cakes using the PF Tek.

As stated before sterilizing the jars, I turned open the lid of each jar a bit.
After sterilization I closed each of them, which caused a major problem while inoculating them in my glove box: when putting the tip of the syringe through the hole (which were covered with duck tape) in the lid it sucked out almost half of the liquid into 1 jar

I am not sure, but probably there was a vacuum of some kind in the jars that caused this, any tips on what I forgot to do? Should I have opened the lids instead of closing them directly. What I did for the other jars is pin the holes with a needle first and that seemed to work...

Any other suggestions? I feel a bit dumb cause of this. Hopefully the other jars will still form mycelium.

 

[Umantis]

My best guess; if the lid was placed on the jar gasket-side down, the jar may have been able to seal even though the band was not tight, even with the duct tape over the prepared holes. The jar would then have negative pressure from the post-sterilization cooling, as in a typical water-bath canning process. Unsure whether this pressure differential alone would be sufficient to account for 5ml of liquid from the syringe, there may be other things going on there.

Another thought, if you didn't flame the needle before underdosing the other jars, possible that the spores will not colonize the substrate fast enough to overwhelm competition from contaminating organisms. Keep an eye on those and be careful to avoid cross-contamination. Good luck!

 

[pitubo]

Obviously, the jars got sealed inside the pressure cooker while it was cooking. Inside the pressure cooker, the jars are bouncing around and the vibrations can twist the lid back on far enough that it effectively seals the jar. When the jar cools after sterilization, it cannot equalize pressure and creates a vacuum inside.

Your can prevent this by putting a small strip of cloth between the jar and the lid. It will prevent the lid from sealing airtight.

After you remove heat from the pressure cooker, put some dense layers of cotton soaked in alcohol over the vent hole of the pressure cooker. When it cools down, it will start sucking back air inside. The cotton will prevent contaminants from entering the pressure cooker and the insides of the jars as they are equalizing pressure. When not only the outside of the pressure cooker, but also the jars inside have cooled down, you can open the pressure cooker and tighten the lid on the jars - after you have pulled the strip of cloth out between the jar and the lid of course.

If you don't trust the trick with the strip of cloth and you have a glove box with clean air, you can twist the lid of the jar just enough for air to enter the jar. That can be very hard to do though with a jar under vacuum, especially within the confines of a glove box... Or if the lid has a hole that was taped over, remove the tape just enough for air to be sucked in and reapply the tape.

 

[EternalPeace]

I don't work with cubensis, but here's how I innoculate my master grain jars:

Just take the lid off in the SAB (still air box), and squeeze a few drops into each corner of the jar. Don't bother with the injection ports, and trying to keep the lids closed all the time, it's really not necessary. Use synthetic filter disks, they work perfectly.

G2G (grain to grain) also--just open the lids inside the SAB, and pour in some white grains.

As long as your being thorough about the disinfection, you should be OK.

Let me know if you need more info, I didn't want to turn this into a wall of text.

 

[Elpo]

G2G (grain to grain) also--just open the lids inside the SAB, and pour in some white grains.

What do you mean by this?

 

[Dead man]

G2G (grain to grain) also--just open the lids inside the SAB, and pour in some white grains.

What do you mean by this?

G2G means transfering part of the inoculated grain into an other partly filled non-inoculated (and sterilised) jar. Thus "grain to grain". If done inside a sab the contamination risk is minimal. This method is ideal for sclerotia producing strains as they hard to fruit and it is thus quite difficult to obtain new spores. It is also great if you want to increase your grow size before your first harvest and don't want to use liquid culture or agar, or simply can't be bothered to collect spores create new syringes etc.
In your case you can just spread the existing jar between 10 new jars and get a much higher yield than just one jar (obviously). If the existing jar is contaminated they will all be contaminated however.
Just make sure you wait until growth is visible to increase chances of success.

Edit: I didn't notice you are using PF, in that case g2g won't work because of the dry layer of vermiculite needed. Sorry! But I hope that at least clarifies things. Assuming you own a pressure cooker I'd suggest looking into grains for your next grow.

 

[Elpo]

G2G (grain to grain) also--just open the lids inside the SAB, and pour in some white grains.

What do you mean by this?

G2G means transfering part of the inoculated grain into an other partly filled non-inoculated (and sterilised) jar. Thus "grain to grain". If done inside a sab the contamination risk is minimal. This method is ideal for sclerotia producing strains as they hard to fruit and it is thus quite difficult to obtain new spores. It is also great if you want to increase your grow size before your first harvest and don't want to use liquid culture or agar, or simply can't be bothered to collect spores create new syringes etc.
In your case you can just spread the existing jar between 10 new jars and get a much higher yield than just one jar (obviously). If the existing jar is contaminated they will all be contaminated however.
Just make sure you wait until growth is visible to increase chances of success.

Edit: I didn't notice you are using PF, in that case g2g won't work because of the dry layer of vermiculite needed. Sorry! But I hope that at least clarifies things. Assuming you own a pressure cooker I'd suggest looking into grains for your next grow.

Okay, very interesting. I was wondering in what stage that needs to be done? Because before the fruiting I will have the chance to take off the layer of vermiculite, couldn't I take a piece of the inoculated grain then to place in a new jar and sterilise after that?

Or maybe just take the vermiculite off inside the sab, and then directly place a grain inside a new already sterilised jar?

 

[Dead man]

G2G (grain to grain) also--just open the lids inside the SAB, and pour in some white grains.

What do you mean by this?

G2G means transfering part of the inoculated grain into an other partly filled non-inoculated (and sterilised) jar. Thus "grain to grain". If done inside a sab the contamination risk is minimal. This method is ideal for sclerotia producing strains as they hard to fruit and it is thus quite difficult to obtain new spores. It is also great if you want to increase your grow size before your first harvest and don't want to use liquid culture or agar, or simply can't be bothered to collect spores create new syringes etc.
In your case you can just spread the existing jar between 10 new jars and get a much higher yield than just one jar (obviously). If the existing jar is contaminated they will all be contaminated however.
Just make sure you wait until growth is visible to increase chances of success.

Edit: I didn't notice you are using PF, in that case g2g won't work because of the dry layer of vermiculite needed. Sorry! But I hope that at least clarifies things. Assuming you own a pressure cooker I'd suggest looking into grains for your next grow.

Okay, very interesting. I was wondering in what stage that needs to be done? Because before the fruiting I will have the chance to take off the layer of vermiculite, couldn't I take a piece of the inoculated grain then to place in a new jar and sterilised after that?

Or maybe just take the vermiculite off inside the sab, and then directly place a grain inside a new already sterilised jar?

I have no experience with pf tek so I don't know I'm afraid. I suggest you check out/search shroomery as there a lor more people that can help you out. The main reason grains are used is that the substrate can be easily broken up by shaking the jar. With pf you could use sterilised tweezers or just clean gloves to break it up perhaps? If you are transferring to sterilised grain I don't see a reason it wouldn't work. Just make sure you break it up good to speed up colonisation.
Also just to make it clear: don't sterilise the jars after you transfered the pf substrate into the grain as that will kill the mycelium! Just sterilise the grain you are transferring to before, the pf should already be sterile.

 

[SynKyd]

Get an account on shroomery, post in cultivation section with pictures. 80% of the Mushroom advice given in this forum is out dated or just dead wrong.

Cheers!

:thumb_up:

 

[Dead man]

Get an account on shroomery, post in cultivation section with pictures. 80% of the Mushroom advice given in this forum is out dated or just dead wrong.

Cheers!

:thumb_up:

The majority of post on shroomery are also outdated or dead wrong . But I agree, as a source of information shroomery is infinitely better if you do your homework and has a lot more knowledgeable members (we're dmt heads not shroom heads here after all).

 

[Elpo]

I already have an account on the Shroomery, but I prefer posting here. From my experience I have always been helped on the Nexus when it came to mushroom cultivation. But of course it couldn't hurt to get a second opinion.

Have been looking up the g2g technique, is quite interesting for future reference.

Thanks :thumb_up:

 

[EternalPeace]

Glad I could help. The concept of G2G is very useful for multiplying your work from a known-good source. Not sure how useful it will be for PF Tek, but when you're ready to move on to other methods, you'll know what to do.