Where'd I go wrong?

[lewiscarroll]

Hi all,

My first attempt at PF tek has been disappointing but by no means a total failure...yet. I started off with 10 jars 11 days ago. Only one inoculation point in one of the jars took. None of the other jars have anything growing in them. I used 3 different syringes with different strains. The jar that is growing looks great so far.

I did another 10 jars 3 days ago using a 4th syringe and also 2 of the original strains - I'll post in a few days what, if anything, is happening with those.

Should I be encouraged yet that there are no contams in the jars that aren't growing? Or do they take a while to manifest?

I used "horticultural" verm which seemed kinda coarse but I think it's ok. The jars had 4 holes covered with micropore tape. I used the steam method to sterilize for 90 min. I built a GB, sprayed it with bleach solution, waited 10 minutes, then wiped it out and wiped it with alcohol, then sprayed oust inside. I cleaned my gloves with alcohol, wiped the tops of the jars with alcohol (this ok?), & flame sterilized the needles between jars. I feel fairly confident my technique was good.

Did I just get bad syringes? Why did I only get 1 out of 40 inoculation points to grow - especially using different syringes from a supposedly good vendor?

Thanks for any help

[teesus]

did you let the needle cool after flame sterilisation? mabe if it was still to hot you might have cooked the spores while injecting.

[Rafe]

Like Teesus said, did you let the needle cool? Did you let the alcohol on the jar lids dry? Did you shake the spore syringes vigorously and often? What temp are you incubating the jars at? Are you keeping them in the dark? Are you taking them out to look at them several times a day? When you sprayed bleach were the foil lids still on the tops? (If not, it could be possible that the bleach got into the holes in the jar) Perhaps the jars are too wet/too dry?

Don't be discouraged and don't give up, the great folks here at Mycotopia will help you figure out what went wrong and have you successful in no time They did for me

[lewiscarroll]

Well, I remember seeing somewhere that the first few drops of solution would more than cool the needle so I didn't wait more than a few seconds. Since I'm using 1cc/jar wouldn't at least some of it worked on each jar?

The alcohol was 91% (which evaporates quicker but doesn't saturate as well as 70% according to something I read here) but I don't really know how dry the lids were - although I wiped them all first, then started injecting so I would think the ones near the end would have been dry for sure if that were a problem. It is slightly humid in the GB because of the oust. But can that much alcohol kill that much solution?

I did shake the syringes a lot & repeatedly.

I sanitized the GB with bleach solution before putting the jars in.

I'm incubating them at 78-80 degrees in the dark.

[Hippie3]

no contams is good-
just give it more time,
spores 'hatch' at different times,
can take over a week sometimes 2.

did you shake syringes well before use ?
spores tend to settle out of solution
and stick to the plastic syringe body-
leaving you injecting water.

how much did you shoot into each hole ?

[teesus]

Originally Posted by lewiscarroll

Well, I remember seeing somewhere that the first few drops of solution would more than cool the needle so I didn't wait more than a few seconds. Since I'm using 1cc/jar wouldn't at least some of it worked on each jar?

i guess this is probably right and there should some spores make it if you shoot 1cc in at once. it was one of my first mistakes when i tryed growing with syringes, so it came in my mind, but i was using only a few drops per innoc hole and flamed the needle between each hole.

[TVCasualty]

Try making a liquid culture jar or two. It'll stretch your spores a long way and give you visual confirmation that you are injecting live mycelium into the jars; if they don't take after that you know something is going on with the syringe or jar and can rule out the spores.

And it being Summer, if you received the syringes recently, did they spend much time in a blistering-hot mailbox?

[lewiscarroll]

Thanks for all your help - the spores did not sit in a hot mailbox, but I have no idea how long they sat in a hot ups truck.

My second batch of 10 jars has 2 of the strain that worked from the first batch and those 2 jars show myc growth as of today. So I now have 3 jars of the same strain going and a bunch of dead jars.

I will try the LC suggestion - thanks! I will have to read up on it first - hope i don't need a PC.

[Hippie3]

well any growth shows that spores are / were in the syringes
so you know that much,
they aren't tapwater
and they aren't full of contams.

[kcmoxtractor]

Originally Posted by lewiscarroll

Thanks for all your help - the spores did not sit in a hot mailbox, but I have no idea how long they sat in a hot ups truck.

My second batch of 10 jars has 2 of the strain that worked from the first batch and those 2 jars show myc growth as of today. So I now have 3 jars of the same strain going and a bunch of dead jars.

I will try the LC suggestion - thanks! I will have to read up on it first - hope i don't need a PC.

You can use Irishlion's tek on LC which just requires you heating it in the oven.
Check it out here ---> http://forums.mycotopia.net/fungi-gr...-syrup-lc.html (Honey/corn syrup LC)

[lewiscarroll]

Well, at least for that syringe. Only 1 out of 4 did anything so far. I'm still wondering if I did something wrong with the alcohol or needle cooling. Any rules of thumb regarding those you might share?

[TVCasualty]

Originally Posted by lewiscarroll

Well, at least for that syringe. Only 1 out of 4 did anything so far. I'm still wondering if I did something wrong with the alcohol or needle cooling. Any rules of thumb regarding those you might share?

I flame the needle once at the beginning of the inoculation session and wipe it with a paper towel soaked in isopropyl, then in between each jar I just wipe it with the towel. I might not even need to flame it the first time, but old habits die hard and it does make it certain to be clean.

Originally Posted by Hippie3

well any growth shows that spores are / were in the syringes

One spot of growth on one jar shows me there's only one syringe (out of three) that has at least a couple of viable spores in it.

My thought was that if 2 of 3 LC's showed no growth and the 3rd showed very slow growth from very few points that it would point to possible accidental sterilization, either when flaming the needle or by sitting in a hot mailbox, and the lack of contamination supports this theory IMO.


lewiscarroll: Though not an issue with this grow, contamination will pop up at some point and that's another huge advantage of doing LC's: If a syringe is contaminated or the spores were killed you only waste a jar of dilluted corn syrup or whatever instead of however many jars you would've otherwise inoculated. And mixing up BRF jars right is trickier than making liquid culture, so you'll looking for ways to store all your surplus fungi soon enough!

[GadgetGuy]

When doing PF tek I wipe the needle with alcohol between every jar and it causes no problems. I will also sometimes have a little bit of alcohol still on the lid and it causes no problems. I flame sterilize the needle before doing a dozen jars and squirt a tiny bit out due to liquid sitting in the needle cover. Use that 1 good syringe to make an LC you will never look back. A person can operate off of one spore syringe for a long time by making lc's. Especially if they keep making new lcs from the ones they have already made.

[cattlexing]

bunk advice deleted

[lewiscarroll]

Originally Posted by GadgetGuy

When doing PF tek I wipe the needle with alcohol between every jar and it causes no problems. I will also sometimes have a little bit of alcohol still on the lid and it causes no problems. I flame sterilize the needle before doing a dozen jars and squirt a tiny bit out due to liquid sitting in the needle cover. Use that 1 good syringe to make an LC you will never look back. A person can operate off of one spore syringe for a long time by making lc's. Especially if they keep making new lcs from the ones they have already made.

I've been flame sterilizing between jars to keep from cross contaminating. Maybe too much heat is the problem. I'm going to give the LC a go but I'm still reading up on different ways to do it.

[Hippie3]

i see some real bad advice here-
plz do not pass off bullshit as fact.

break it up with your dirty dirt covered hands. Contams and sterelization are not a concern at this point... you got soil on your side.

fyi
only some bacteria are anaerobic - not all, and not molds.
there is no one kind of soil-
there are many, many kinds
and many are NOT suitable for our use.

[cattlexing]

Anyways- One day we should start a thread about soil to have a discussion about it so we can all learn about it, via discussion, in an internet forum.

One question I have is: Is it possible for a contaminate to live inside healthy mycelium.. And is it possibly for the contaminate to deliver anything toxic/allergenic/dangerous through the mycelium.. into the mushroom. If so... i don't see why we eat shrooms off the cow pattie by the ton. Just wondering...

[Hippie3]

well, worms for one. and some molds create toxins as by-products. but for the most part one can pick and eat right off the ground if hygiene isn't any concern.

[TVCasualty]

Originally Posted by cattlexing

One question I have is: Is it possible for a contaminate to live inside healthy mycelium.. And is it possibly for the contaminate to deliver anything toxic/allergenic/dangerous through the mycelium.. into the mushroom. If so... i don't see why we eat shrooms off the cow pattie by the ton. Just wondering...

By 'live inside' do you mean within the cell walls of the mycelium itself or just really close to or touching the mycelium but outside the actual cells?


Some viruses can contaminate and live inside mushroom tissue but they generally are harmful to the mycelium or prevent any fruiting. Another issue to consider is that many mushrooms are proven to be hyperaccumulators of heavy metals in the soil, so I would never eat a mushroom picked from a probable toxic waste site, and since those are often hard to spot I don't eat that many wild mushrooms because I don't live close to any decent wilderness areas far from possible industrial contamination.

Regarding wild- or pasture-picked mushrooms I'm a lot more concerned about things like chemicals and heavy metals than bacteria, fungi, viruses, or worms because I'd be either cooking the mushrooms or drying them.

It's really not a good idea to eat fresh mushrooms off cow patties. At least make some tea or dry them first or something. That said, lots of people have done it (including me on occasion) without any horrible things happening to us. Others have done it and regretted it. Read/learn as much as you can and use your best judgment.

That's about as far off lewiscarroll's topic as I think we should go in this thread, so for a primer in basic microbiology it's best if you started a new thread.