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Liquid culture experiment....Hillbilly's syringe refill kits...strange results


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#1 BuckarooBanzai

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Posted 12 October 2005 - 09:46 PM

Okay, so my FOAF got one of Hillbilly's syringe refill kits:

http://mycotopia.net...light=hillbilly

He HIGHLY recomends them, by the way. Since my friend had so much primo spore water, he decided to do some LC experiments. Basically, he wanted to compare growth properties of Karo and Dextrose in vented and unvented jars.

So, he prepared 8 jars. Four with 4x layers of tyvek (separated by/siliconed to thick washers) and four without filters. All eight jars had one silicone air port and three glass marbles (to aid in swirling). The sugar recipe was 1sp dex/Karo per 100mL of distilled water. All eight jars were set aside for 24hrs and were then PCed at 15psi for 30m. All eight jars were cooked with the seal side down.

Each jar was innoculated with 1/4cc of spore water. The non filtered jars were innoculated carefully, because each contained a vacuum and my friend didn't want to loose a whole syringe in each one. The filtered jars were just squirted.

Now on the evening of day four they are all showing nice growth and no hint of contamination. Here is the strange part:

The four jars without filters (the vacuum jars) are at least twice as colonized as the filtered jars. The vacuum jars germinated/showed first and graduated to big puffballs of mycelia first. The vacuum puffballs are much larger than the filtered puffballs. These general results are pretty constant across all the jars. Even the slowest of the vacuum jars is doing far better than the fastest of the filtered jars.

I've been whacking my head against the net all evening trying to find some kind of reason why spores/mycelium under vacuum would grow more quickly than under standard pressure but thus far I've found nada. If I had some clue why this was going on, I could begin cogitating on how to exploit this effect.

Anybody else seen anything like this? Unless the vacuum jars stall massively, there is no way the filtered jars could possibly hope to catch up. Perhaps I've got a contamination issue that isn't yet visually obvious, but in all four filtered jars?

On the actual experimental front, the Karo and Dextrose seem pretty much equivalent. Dextrose showed a few hours before the Karo, but everything seems pretty much equal at this point.

Hobson, your thoughts?

:eusa_thin

#2 Hippie3

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Posted 13 October 2005 - 09:06 AM

perhaps the explanation is the low pressure.
it seems possible that the mycellia could just APPEAR larger
if the pressure on it was reduced, as in a partial vacuum.
the total tissue weight would be the real determining datum.
dextrose = karo, i.e. corn sugar

hillbilly's kits rock, eh ?
i agree.

Each jar was innoculated with 1/4cc of spore water.


lemme see,
that would be about 40 jars of LC per syringe,
you get ~ 20 syringes from hillbilly's kit,
that would make up 800 LC jars
each holding about another 20 syringe-fulls
for a theoretical grand total of
~ 16,000 syringes. all for an investment of about $25-35
including extra jars, karo, etc.

the spore concentration in hillbilly's kits must be quite high
to get such impressive growth so quickly from such a small amount.
:headbang:

#3 BuckarooBanzai

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Posted 13 October 2005 - 07:24 PM

Dude, I could EASILY dilute that water 5 to 1 and still have good syringes (I might just put that to the test, actually). Hillbilly claimed it was only one print in the jar. If that's the truth, that print must have been about 5 inches across! I am totally psyched to have such a large supply of water to work with as a "control." I know there will be genetic variation from spore to spore, but not as much as there would be in five different syringes from five different sources!

Jar growth has been explosive, as well. Potent water and clearly VERY fresh from the aggressive colonization speeds. Also, since the water is in a big jar, it's easy to shake up and homogenize before inoculation without having to resort to surfactants/ultrasonic’s. Since I'm experimenting here, having zero additives I don't know about is absolutely essential. I don't just want answers, I want REPRODUCABLE answers. Any vector I can't reproduce effectively wrecks my whole process.

By the way, your Mycrotopia substrate is pretty effectively beating the standard brown rice flour formula in colonization speed. I hope those results translate to Psilocybin content, as well! What I would give to have access to gas chromatography equipment...

I had considered the difference in growth being a purely visual phenomena based on the low pressure...at first. At this point, though, I don't just have much larger puffballs, I've got FAR more of them in the vacuum jars (wish I had a camera that shot good closeups). I had not considered a dry weight test - but it's an excellent idea! Numbers don't lie, eh? I'll keep two of each jar type for a weight test at the end. Eight jars of mycelium is far more than I have the facilities to exploit anyway!

As to the Karo being the same as dextrose...not quite true. Karo is a mixture of light and high fructose corn syrups plus vanilla and preservatives. My point with the experiment was to determine if the “off the shelf” mix of sugars (plus vanilla) would be any more or less effective than pure (lab grade) anhydrous dextrose. I can most assuredly tell you which one is cheaper and easier to get!

The next round of tests will be dex vs. malt enriched dex, followed by dex vs. potato enriched dex. The most successful dex solution will then be tested with/without a yeast addition. I'm very interested in seeing if what I already know about agar based nutrients translates to liquid culture nutrients. There just aren't enough folks out there publishing hard numbers in this arena...and his eyes glaze over as he fantasizes about scribbling in his little book leading to a "discovery"...

Incidentally, based on the speed the mycelium has ripped through these jars, the next rounds will be done with 1/8cc of spore water per jar. Waste not, want not, you know? For those of you doing the math, that would be around 30,000 mycelium syringes from one jar of water. All while supporting this fine institution. What more could one ask?


#4 Hippie3

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Posted 14 October 2005 - 06:20 AM

your Mycrotopia substrate is pretty effectively beating the standard brown rice flour formula in colonization speed.


:eusa_danc :eusa_danc :eusa_danc

yep, my latest formulation kicks ass,
best to date.

btw, i'd refrigerate the spore kit when not in use
else you may see premie germination in it.


lazlo seems to think that vanilla helps growth some
so perhaps its' presence in the karo is significant.
but it also seems possible that the lower pressure might
be the factor allowing formation of more myc. clouds,
perhaps rendering visible ones that under higher pressure
might be too small to see yet.
so weight will be the real test.
interesting work you are doing here,
keep us posted plz.
thx for sharing
:bow:

#5 Hillbilly

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Posted 14 October 2005 - 06:36 AM

Hillbilly claimed it was only one print in the jar. If that's the truth, that print must have been about 5 inches across!


The prints I use are about 3 inches across but I do use ultrasound to break up the spore clumps, making it seem like there is more in the jar. I'm very glad you are happy with my kit, just doing my part to support this great website.

#6 BuckarooBanzai

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Posted 14 October 2005 - 08:05 AM

Hillbilly: I am more than just happy with your kit, I am ecstatic! My original plan was to cough up the dough for five syringes and mix them up so I could have a decent "control" source to experiment with (getting more than five syringes, from the same print, was far more than I had hoped for). Fruits are cool, but playing with variables and recording results is cool too (please note, I am an utterly unrepentant GEEK when it comes to science).

Hippie: My house is usually in the 68F-70F range (me and 'da wife like it chilly). Do you think the fridge is still a good idea?

Also, I've seen some wildly different opinions on how long spore water will remain viable. What do you guys think is the outside maximum while still maintaining a good degree of vigor?

Finally, I can't find any hard data on freezing Psilly spores in water. Any thoughts?

:bow: THANKS SO MUCH ! ! ! ! :bow:

#7 Hippie3

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Posted 14 October 2005 - 10:52 AM

i'd still refrigerate, your solution will last much longer if you can prevent premie germination as once the spores germinate they will begin cellular divisions [aging process] and they will need food, etc.
better to keep 'em dormant until ready to use.
but don't freeze them,
a long hard freeze will kill the vast majority.
vigor in terms of germination starts dropping after about a year,
by the end of year two it's significant enough to make a difference.

#8 Hippie3

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Posted 14 October 2005 - 11:07 AM

Dude, I could EASILY dilute that water 5 to 1 and still have good syringes (I might just put that to the test, actually).


that too would be an interesting experiment.

#9 BuckarooBanzai

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Posted 15 October 2005 - 01:21 AM

1 week update:

The Karo jars seem to be pulling ahead slightly. Also, the Karo growth is more "fluffy." The puffballs of mycelia in the Karo jars have much softer edges, less obvious divisions/seperations. Also, based on a few very large clouds, it seems that the Karo myc is pulling together. Dextrose jars are showing much more defined spheres.

Vacuum jars are still way, way ahead. The best development, visually, is a Karo/vacuum jar. The worst, by far, is a vented dextrose jar.

Damn glad I didn't pay anything for that anhydrous dextrose...

#10 Hippie3

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Posted 15 October 2005 - 05:30 AM

any pix ?

#11 shobimono

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Posted 15 October 2005 - 06:42 AM

1 week update:

The Karo jars seem to be pulling ahead slightly. Also, the Karo growth is more "fluffy." The puffballs of mycelia in the Karo jars have much softer edges, less obvious divisions/seperations. Also, based on a few very large clouds, it seems that the Karo myc is pulling together. Dextrose jars are showing much more defined spheres.

Vacuum jars are still way, way ahead. The best development, visually, is a Karo/vacuum jar. The worst, by far, is a vented dextrose jar.

Damn glad I didn't pay anything for that anhydrous dextrose...


One thing to keep in mind is that there are definately 2 substrains in the Hillbilly strain. One grows short, thick texas like fruit and the other grows very tall sturdy large capped fruit.
This could account for part of the different growth patterns and rates you are seeing between the different jars.
Foaf is experimenting with an lc of karo/starch/nuteyeast and she has noticed different growth patterns based on different strains and species.
Redboy is growing in a definate spherical shape, while PF Albino is growing in a more whispy shape. The various Pans she is trying have a real spikey rhizo look to them. Atlantis #7 looks spikey too.
The lc's she is using are also unvented. She finds that works better than a vented lc. The problem she has found with a vented lc is that the water slowly evaporates, thus concentrating the nutrient level in the jar which slows or stalls growth.

#12 BuckarooBanzai

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Posted 15 October 2005 - 11:23 AM

Hippie3: Sorry, no pics. At least no ability to take the close ups that would actually be of any use to our discussion here. Want to see my jars from four feet away? Brings up a question I had been intending to ask: is there a thread out there on inexpensive digital cameras that take good close up shots? Some of the folks on this board are taking beautiful pics, I would love to know what hardware they are using...I know jack about cameras. Also, I wanted to spend those initial big bucks on a 22qt pressure cooker and my HEPA/blower for my ghetto flow hood!


Shobimono: It was my understanding that at this early stage, mycelial growth should be pretty much universal in appearance. That is clearly not your experience. Perhaps I have drastically overestimated the "control" nature of my spore source. Will rhizomorphic character be visible this early?

Once I have fruit, I think I will rerun this process with cloned tissue. Have you noted variance in mycelial growth within a cloned subset? I remember something about folks having noted sectoring in clone tissue, so perhaps that's not a way to go either.

I have to question the substrain theory, though. The variance in pattern and appearance of growth is constant from jar to jar. All the growth in the Karo jars is fluffier whereas all the growth in the dextrose jars is more spherical. All the vacuum jars are ahead of the vented jars. My cross section is small enough, though, that I could easily have two substrains split up amongst the eight jars equally. It would be a statistical aberration, but not a massive one.

I hadn't even CONSIDERED evaporative concentration and the subsequent nutrient imbalance (tunnel vision on fresh air/gas exchange). A most excellent point to consider that I thank you for brining it up! Perhaps profiling sugar contents would offer some value here. Benedict's reagent is pretty cheap...something to add to the wish list!


Hillbilly: I'm dense, yes, but I just "got" your avatar! Weather station...LMFAO!!!!!!!!!

#13 Hippie3

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Posted 16 October 2005 - 05:52 AM

a 5 megpxl cam should do you right, nikkon or canon are my favs

#14 BuckarooBanzai

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Posted 16 October 2005 - 06:37 PM

Looks like you was right, boss.

I pulled my spore water out tonite to do some more work and....some little tiny white threads inside where there were none before. I should have tossed them in the 'fridge the minute I read your post but no, I had to play little league...

My ASSUMPTION: It's nothing really to worry about. The tiny amount of available nutes will be consumed quickly and put the new myc into suspended animation shortly after hatching (and all the spores won't hatch anyway). Not so bad, in fact, because work done from this jar from now on will be live cuture work and take off faster.

Only downside I see: it's going to be vigorous for a shorter time, even in the fridge.

These are the ASSUMPTIONS of a total newbie. Please correct me as requisite.

Oh ye of experience, how best to proceed to maintain vigor for the longest time? Maybe give up on vigor and term storage and throw some nutes straight in the jar?

Do all water prints/syringes germinate invitro? Prints are starting to make more and more sense...

#15 Guest_dial8_*

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Posted 17 October 2005 - 08:30 AM

Very, very nice. Seems like the difference in growth is due to the pressure. Makes sense in theory anyway. The vaccuum jars are under less pressure so the myc "expands" easier.

#16 blackout

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Posted 17 October 2005 - 11:00 AM

Which jars did you do first? If you had spores in a syringe that was held vertically the spores will settle in the bottom and the first few squirts will have more spores. Also did you shake the vacuum jars more? Aeration will lead to better growth. It is a catch 22 with some filter jars, i.e. you cannot shake hard for fear of wetting the filters, so they have less aeration in the liquid.

#17 Hippie3

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Posted 17 October 2005 - 11:15 AM

that's why a silicon adhesive blob is better than any fiber filter,
you can shake the [email protected] out of the LC with impunity

also see
http://mycotopia.net...ad.php?p=194919

#18 Hillbilly

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Posted 18 October 2005 - 02:13 PM

Hillbilly: I'm dense, yes, but I just "got" your avatar! Weather station...LMFAO!!!!!!!!!


Thanks, It's been cold here lately, almost put my eye out.
:)

#19 BuckarooBanzai

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Posted 18 October 2005 - 04:55 PM

I shot the non-filter jars first and then re-filled the syringe. I shot each of the vaccum jars individually because they will suck whatever is in the syringe out. All pulls were from a large Hillbilly jar that had the living crap shaken out of it before each use, so I don't think spore dispersion is the issue.

All my filter jars have 4 layers of tyvek (seperated by 5mm thick washers), so I can shake them without concern of wetting the top filter. Each day, they are given one vigorous 30 sec shake and 4 thirty second swirls.

From another thread (http://mycotopia.net...ad.php?t=5206):
"I think the exact morphology of the mycelium is a complex function of the stir rate, original amount of inoculants, whether it was a spore solution or liquid culture inoculation, type of mycelium, temperature of incubation, length of incubation, the types of sugars used, and other things.

I tend to agree with that theory. I'm going to test it by shooting a dex jar with Karo mycelia and see if I get the different visual appearance from the same material.

The vacuum jars are still very much outperforming the vented jars...must be enough air in there, no sign of stall yet. The real proof will be after I dry weigh the mycelia, though.

Hippie: Damn, dude, your substrate ROCKS! I've got two jars that are %70-%80 colonized (my standard BRFs look WEAK by comparison). You puttin' some steroids in that stuff, or what???

#20 Hippie3

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Posted 18 October 2005 - 06:37 PM

Hippie: Damn, dude, your substrate ROCKS! I've got two jars that are %70-%80 colonized (my standard BRFs look WEAK by comparison). You puttin' some steroids in that stuff, or what???


kinda,
plant growth hormones.
works well, eh ?
:reb:




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