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Offlinenugjug
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Registered: 11/07/05
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A few questions
    #5862624 - 07/15/06 06:45 PM (17 years, 8 months ago)

Right now I am experiencing very slow colonizations on my rye jars and I am just wondering if anyone can provide any insight as to what is causing this problem.

I soaked the grain in quart jars and continuously added water until the grain stopped absorbing water. I then strained it and ran it under the shower for a while in the strainer. I then let it sit for about a half hour to get the excess off. I then put 3 cups in each quart jar, put the lid on and PCed for 90 minutes. Took them out, shook them and let them cool over night. Shot them up the next morning with 1cc of spore solution.They have been sitting in an incubator at around 83 degrees F. It takes 14-18 days before I even see the slightest sign of myc and then about another 16-20 days to finish colonizing. The jars have a quarter inch hole covered in tyvek for gas exchange. Anyone have any thoughts as to what I could be doing wrong?

Additionally I have made two LCs using malt/dextrose per agar's directions. I.e. one table spoon of malt and one table spoon of dextrose per 4 cups run through double coffee filters. Then divided between two quart jars and PCed for around 20 minutes. These always come out with so much malt/dextrose settled at the bottom. I shot them both up with about 1.5cc of spore solution and they are crawling along. One has about 3 pieces the size of a match head floating around and the other one shows zero signs of growth. Did I use to much malt/dextrose? Did I not filter it well enough?

Could all of this be the result of bad syringes?

Sorry for the long ass post. To those of you who suffer through it and have some help thank you in advance.

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Offlinenugjug
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Re: A few questions [Re: nugjug]
    #5865404 - 07/16/06 12:36 PM (17 years, 8 months ago)

Anyone have any thoughts? Is this just characteristics of this strain? I know it is psilocybe cubensis but as far as the actual strain goes I am not sure. I got the prints from a friend who got them from a friend who is no longer in contact.

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OfflineCarbon_Black
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Re: A few questions [Re: nugjug]
    #5865517 - 07/16/06 01:02 PM (17 years, 8 months ago)

Make sure your water content is just right in your jars, you should have no free standing water or droplets, and the grain shouldn't be dry.  That's probably the biggest factor aside from temp and air exchange, both which seem adequate in your case.  For the liquid culture try pc'ing the lc mixture first instead of boiling, then run it through two coffee filters to remove sediment.  Then recook in pc.  Also make sure you aren't overcooking the jars and carmalizing the sugars.  Keep trying and everything will work out for you.
good luck! :thumbup:


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As Sure As The Sun Will Shine

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Invisiblemonstermitch
Growing in Bags Doesn't Work

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Re: A few questions [Re: Carbon_Black]
    #5865572 - 07/16/06 01:32 PM (17 years, 8 months ago)

doesn't sound like any care was taken to germinate
bacterial endospores...

slow growth now..
bacterial contamination is my guess.
due to your lack of soaking and/or simmering.

how about trying again and picking a good tek to follow.
follow it to the T, and you will have no problems.


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InvisibleOptx
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Re: A few questions [Re: monstermitch]
    #5865957 - 07/16/06 03:48 PM (17 years, 8 months ago)

um...he said he soaked...


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please do not take everything i say here so personally. welcome to teh internets!

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Invisiblemonstermitch
Growing in Bags Doesn't Work

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Re: A few questions [Re: Optx]
    #5865974 - 07/16/06 03:51 PM (17 years, 8 months ago)

but for how long???


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InvisibleOptx
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Re: A few questions [Re: nugjug]
    #5865984 - 07/16/06 03:54 PM (17 years, 8 months ago)

Quote:

nugjug said:
...
I soaked the grain in quart jars and continuously added water until the grain stopped absorbing water...




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please do not take everything i say here so personally. welcome to teh internets!

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Invisiblemonstermitch
Growing in Bags Doesn't Work

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Re: A few questions [Re: Optx]
    #5865991 - 07/16/06 03:59 PM (17 years, 8 months ago)

not long enough in my book.
but still could be fine I guess...

but that is only one part that could have gone wrong.
what about the sterile technique?
inoculation technique?

and again 1cc per quart of grain isn't very much at all.
and if he shoke the jars after inoculation, well then
that could really have slowed him down.

I would say use more inoculant, and have it be LC.

are the LC's cloudy at all? Opaque or a thick translucent?


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InvisibleOptx
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Re: A few questions [Re: monstermitch]
    #5866002 - 07/16/06 04:04 PM (17 years, 8 months ago)

yeah. 1 cc is a bit light.
but i've read about lots of people either taking out the 24hour soak or the 30minute simmer and still having good results.


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please do not take everything i say here so personally. welcome to teh internets!

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Invisiblemonstermitch
Growing in Bags Doesn't Work

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Re: A few questions [Re: Optx]
    #5866013 - 07/16/06 04:08 PM (17 years, 8 months ago)

you are correct.

p.s. doesn't do either.
just thinking where the bacteria came from.

most likely, if there even is any, it came from his breath.

and if it takes about 16 days to see mycelium,
but only another 16 days to fully colonize,
that makes no sense.

and really there seems to not really be a problem.
they colonized totally, correct?
32 days or so is not off par for 1cc of spores at all.

the colonization probably started in the center and moved
towards the glass.


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Invisiblemonstermitch
Growing in Bags Doesn't Work

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Re: A few questions [Re: nugjug]
    #5866019 - 07/16/06 04:10 PM (17 years, 8 months ago)

so to speed things up for you...

soak your grains in a weak coffee solution with some
gypsum added to it.
inoculate with liquid culture (if you make a good one)
and use about 5cc's.

shake after colonized about 30% or so.

should speed you up a bit.


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Offlinejcee
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Re: A few questions [Re: monstermitch]
    #5866028 - 07/16/06 04:15 PM (17 years, 8 months ago)

agree with mitch

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Offlinenugjug
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Re: A few questions [Re: jcee]
    #5868605 - 07/17/06 08:50 AM (17 years, 8 months ago)

OK, those were pretty much the thoughts that I was having mitch.

All the inoculations were done in a glove box through self healing injection ports and the soak took over 12 hours.

As far as the liquid culture goes it is cloudy with the malt/dextrose forming a sediment layer at the bottom when allowed to sit. I made it by putting the malt/dextrose in a double layer coffee filter and placing it into a coffee maker and brewed a pot. Then I pressure cooked it. So Carbon_Black you are saying to pressure cook it and then run it through the coffee filters and then pressure cook it again? Also are my measurements correct. One table spoon of each to four cups of water? Also I used tap water, is this a problem?

On the times it was more so a guess as to when they would finish based on growth. I recently shook the jars after they looked about a third of the way done and it is now clear that my estimates were off. This is however the first time I have shaken the jars since the initial shake out of the pressure cooker. Most of the colonization started in the bottom of the jars where the spore solution had settled.

I have been waiting for internet connection which I just got to research the coffee soak a bit further.

Thanks to everyone for their help and to those who will continue to help.

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Invisiblecreamcorn
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Re: A few questions [Re: nugjug]
    #5868617 - 07/17/06 08:58 AM (17 years, 8 months ago)

No need to PC twice. You just need the water warm when you mix your malt/dex so that it dissolves easier, filter like you said w/coffee filters if desired (which isn't even necessary, the sediments don't hurt a thing, it just makes your LC pretty), then PC. I've personally had poor results with tap water, suppose it depends on the quality of your water locally. If in doubt, use spring or distilled water from a bottle.

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Offlinenugjug
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Re: A few questions [Re: creamcorn]
    #5868692 - 07/17/06 09:31 AM (17 years, 8 months ago)

I was just worried that there might be to many nutrients in the water to the point that it some how hinders the spores from germinating. I don't know still trying to figure things out. Plus not being able to see what is happening in the solution is a bit unnerving for someone still trying to get their LCs right. Should I leave it off the magnetic stirrer until I see signs of growth or should I just toss it on there right after i knock it up?

Edited by nugjug (07/17/06 02:12 PM)

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Invisiblecreamcorn
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Re: A few questions [Re: nugjug]
    #5868823 - 07/17/06 10:26 AM (17 years, 8 months ago)

Its very possible that there's too many nutes, and it definitely does hinder growth. Spores won't germinate at all if your liquid is too "dense" with sugars - its due to osmotic pressure I believe, the liquid becomes too "thick" that it exerts a pressure on the spores, rendering them unable to germinate. I don't feel like doing the math for your numbers but you want roughly 4%. I do it by weight, 1ml weighs 1 gram, so I measure 4 grams of media per 100ml of water. When in doubt, less is usually better to be on the safe side.

Leave it off the mag stirrer until you see signs of growth. Remember that a germinated spore is monokaryotic, and can not fruit until it mates with a compatible spore (dikaryotic). (Of course if you some how end up with nothing but monokaryotic mycelium in your culture, they can still meet and mate once inoculated...) This is difficult to do in an LC in the first place, but luckily spores clump up and I'd wager they do mate. I'd leave it totally undisturbed for the first few days, once you got a little cloud going you can start mag stirring it.

Edited by creamcorn (07/17/06 10:33 AM)

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Offlinenugjug
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Re: A few questions [Re: creamcorn]
    #5869161 - 07/17/06 12:25 PM (17 years, 8 months ago)

Thanks creamcorn. I was going off of agar's recipe but just did the math to check. 4 cups is 64 tablespoons. So would that be 2/64 or 2/66? Either way it comes out to less than 4 percent.

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InvisibleTippinthru
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Re: A few questions [Re: nugjug]
    #5869204 - 07/17/06 12:39 PM (17 years, 8 months ago)

Quote:

nugjug said:
Thanks creamcorn.  I was going off of agar's recipe but just did the math to check.  4 cups is 64 tablespoons.  So would that be 2/64 or 2/66?  Either way it comes out to less than 4 percent.





agar's recipe worked perfectly, for me :grin:


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Offlinenugjug
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Re: A few questions [Re: Tippinthru]
    #5869430 - 07/17/06 02:15 PM (17 years, 8 months ago)

Trippinthru did you PC then run through coffee filters and then re-PC as he suggests? When I tried my first time I did this and ended up making a mess trying to pour through the double coffee filters. If you do PC once, filter and then PC again do you run your's through an actual coffee pot after you initially PC or pour it somehow?

Thanks to all for any help.

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Invisiblebaycafe
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Re: A few questions [Re: nugjug]
    #5869476 - 07/17/06 02:30 PM (17 years, 8 months ago)

Last time through I wrapped a rubber band around the filters over the mouth of the jar. The filter should still be concave just like you might expect a coffee filter to look when in a coffee maker. This free up your hands and lessens the mess, just make sure to filter slowly.


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I think my eyes are getting better. Instead of a big dark blur I see a big light blur.

俺のシロシャイビケッルリプスがここです。

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