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Invisiblewise_gardener
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Registered: 08/02/05
Posts: 157
Please help me determine what is my contamination vector!
    #4813539 - 10/16/05 10:26 PM (18 years, 5 months ago)

Hi, (please excuse the plain english)
I give another try and start a new psilocybe cultivation and again, have little troubles. Here exactly how I proceed :

Grain used : Organic rye grain
Spore used : Psilocybe cubensis Mazatapec from Sporeworks 10cc syringe
Jar used : Pint/500 ml. jar

  1. I first soak the rye grain 30 hours at room temp, to let the grain absorb all possible moisture and let bacteria endospore germinate.
  2. I put the grain in a spaghetti drainer and rinse well.
  3. I let the grain drain well in this container for about 5 minutes.
  4. I load the pre-washed jar to 75% of their capacity with the prepared grain.
  5. I put a Tyvek lid over the jar, and close firmly with jar tops.
  6. I covered each jar with 2 foil lid.
  7. I put 1 inch of water in the pre-washed pressure cooker and put the jar inside.
  8. I close it, increase the heat gradually, and let the steam come out of the PC 5 minutes without the pressure gauge.
  9. I then put the pressure gauge, and pressure cook 1h15 at 15 psi.
  10. Within this time, I clean well all my open air ?laboratory?.
  11. Once the sterilization process is complete, I bring the PC to my laboratory and let the pressure drop down.
  12. Once all the pressure is discharge, I open the PC, take a laundry, and shake all my jar to well distribute dry and moist grain.
  13. I then put a clean clothe over the jars and let them cool down overnight (about 12 hours).
  14. When the jar is at room temp, I clean all the working surface. Now I take a shower, wear clean clothe, and return to my laboratory.
  15. Now I close all doors, windows and computer/fan to minimize air circulation.
  16. I then clean my hand with anti-bacterial soap, take my syringe from the refrigerator, et prepare the tools for the inoculation. I then re-clean the working surface, and my hand.
  17. Now I take a lysol spay bottle and spray a bunch of lysol all over the air, to sterilize it as much as possible.
  18. I then put a surgical mask and re-clean my hand.
  19. I now take the clothe over the jars off, and shake the syringe well. (syringe already have air inside so I don?t need to suck some trough fire)
  20. I now take off the 2 foil lid from the jar. Here I have noticed a little bit of water over the tyvek. Some water seem to get stuck between the first foil lid and the tyvek sheet.
  21. I then prepare a scott towel sheet imbibe off 90% isopropyl alcohol. I take the syringe needle protector off, and burn the needle red hot to sterilize it. I then let the needle cool down in the isopropyl alcohol sheet.
  22. I approach much as I can from the jar with the needle inside the alcohol scott towel, and inject 1cc. of solution in the middle of the jar.
  23. Now I seal the little syringe hole with 3m surgical tape. Finally shake the jar to disperse spore solution and put it in the incubator at 27-28c.
  24. I repeat the 2 previous steps for each jar. Now look at the jar 4 days after inoculation :





It's a little bit difficult to see but there some greenish spot on some mycelium growth! What the hell am I doing wrong ? It's pretty frustrating. I read so many teks and know how to do from the begining to the end, and can't even pass trough the grain colonization step  :mad2: :mad2: :mad2:.. Please help me if you see something wrong in my procedure!

thanks a lot..
wise_gardener

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OfflineMycophilic
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Registered: 09/17/05
Posts: 104
Loc: WA, USA
Last seen: 14 years, 2 months
Re: Please help me determine what is my contamination vector! [Re: wise_gardener]
    #4813896 - 10/16/05 11:18 PM (18 years, 5 months ago)

dirty syringe? it wouldn't hurt to pressure cook for 15 more minutes either

Edited by Mycophilic (10/16/05 11:19 PM)

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Invisiblewise_gardener
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Registered: 08/02/05
Posts: 157
Re: Please help me determine what is my contamination vector! [Re: Mycophilic]
    #4815208 - 10/17/05 08:09 AM (18 years, 5 months ago)

any other ideas ?

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Invisiblebackupwards
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Registered: 04/02/05
Posts: 3,022
Loc: somewhere else
Re: Please help me determine what is my contamination vector! [Re: wise_gardener]
    #4815227 - 10/17/05 08:25 AM (18 years, 5 months ago)

i could be wrong here but they look entirely too wet. maybe not though.

1hr 15 minutes should be plenty of pc time, don't see a need to go more than that. all your procedures seem to be fine. here is where i think your problem may be coming from: once the tyvek gets wet it will allow contams to work their way through. since you are inoculating and then shaking to distribute spores, are you making sure not to get the tyvek wet. you say there was water on it when you took the foil off, this would tell me that you don't have the foil pressed over the lid well enough or you have too much water in the pc to start. an inch or two up the sides of the jar is all you should need.
also after you place the rocker on the pc and the steam starts coming out, do you turn down the pc so the rocker just moves a little bit, you don't want it blasting steam out all the time.
:peace:

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Offlinefrank_grimes
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Registered: 10/12/01
Posts: 277
Loc: Springfield, USA
Last seen: 1 year, 4 months
Re: Please help me determine what is my contamination vector! [Re: wise_gardener]
    #4815247 - 10/17/05 08:31 AM (18 years, 5 months ago)

Quote:

wise_gardener said:
any other ideas ?




The way I look at this hobby is that loss is part of the game. I expect between 10-20% loss due to contamination. I have worked in very dirty environments and gotten great results, I have worked in almost perfect conditions and gotten bad results. The poster above makes a good point about the wet tyvek, but no matter how clean you try to be, contamination happens.

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Invisiblewise_gardener
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Registered: 08/02/05
Posts: 157
Re: Please help me determine what is my contamination vector! [Re: backupwards]
    #4815320 - 10/17/05 09:00 AM (18 years, 5 months ago)

thanks!
I'll try to be more careful next time when I'll put my foil sheet.. Normally I only press on the side of the aluminum sheet/jar, not on the top! I did not know that this is necessary. What's a stupid error anyway! I only have 1 syringe left on a 4 lot, chitwan, please be my revelation...

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OfflineMycophilic
thrill seeker
Registered: 09/17/05
Posts: 104
Loc: WA, USA
Last seen: 14 years, 2 months
Re: Please help me determine what is my contamination vector! [Re: wise_gardener]
    #4815594 - 10/17/05 10:43 AM (18 years, 5 months ago)

and if you have loss make sure you do outdoor inoculations

some folks say that it might be better to dry the grain flat rather than in a round drainer too, although i've never tried that i just let the grain dry after soaking for an hour, maybe

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Invisiblewise_gardener
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Registered: 08/02/05
Posts: 157
Re: Please help me determine what is my contamination vector! [Re: wise_gardener]
    #4815614 - 10/17/05 10:48 AM (18 years, 5 months ago)

About this, is it harder to colonize rye than other grain substrate ? WBS for example. I'm at my third try with no success and definitely want to know what I'm doing wrong. First try, contams, second try, mycelium growth stop, and third try, contams.

It would be a waste of potential great shrooms! Look at my backward...




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