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Offlineurthtown
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slow growth shiitake
    #19259690 - 12/11/13 11:30 AM (10 years, 1 month ago)

Hello folks, I took a tissue sample from a store-bought and healthy looking shiitake to practice my agar technique in my SAB. A few practice runs before my cultures and PC arrive.

This has been on agar just over a week, does this growth seem slow to you? I've read several posts by RR that commercially available edibles have probably been grown out through G2G so much that the culture is close to senescence by the time it is introduced to fruiting conditions. I can't remember which post I saw him comment on but he pretty much said that a tissue sample from such a fruit would probably produce a bit of myc that would spread slowly on the agar and then just stop. Does this look like what is going on with my practice clone here? Elderly tissue sample just can't get his/her growth on?



It has grown a little bit since that pic, I didn't want to open it again so this pic just shows that it's getting to be a stronger white.



In a couple weeks I'll have a master culture on a petri and I'll grow out from there but for now just curious and still learning. Thanks!


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Edited by urthtown (12/11/13 11:40 AM)


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OfflineAbbeyRoad
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Re: slow growth shiitake [Re: urthtown]
    #19260820 - 12/11/13 03:21 PM (10 years, 1 month ago)

What kind of a lid are you using?  Also the agar looks like its puddled from condensation which will definitely slow growth and encourage bacterial contam.  I never crack a colonizing dish until I'm ready to do the respective transfer.


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Offlineurthtown
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Re: slow growth shiitake [Re: AbbeyRoad]
    #19261945 - 12/11/13 06:48 PM (10 years, 1 month ago)

It seems like all my jars of agar have had condensation that has run down and puddled on the surface. How do you prevent this? I haven't seen specific reference in any agar teks to preventing condensation.

The lid is just an upside down wide mouth mason jar lid. I cracked it to take the photo because this isn't meant to be a culture I grow out really, just to practice my technique and learn what I can.

I even cracked a jar of each batch of agar for a few seconds in my SAB and then put the lid back on to track contamination due to technique against a sterilized plate that was never opened. So far things are staying pretty contam free...

I might practice a transfer with this myc onto a clean plate also as practice and just see what happens. My PC arrives on Friday  :evil:


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Offlineurthtown
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Re: slow growth shiitake [Re: urthtown]
    #19261959 - 12/11/13 06:50 PM (10 years, 1 month ago)

I should say also, I've built lids with SFD sir ports and silicone injection ports for work with agar and grain jars once I have my PC - these test dishes were just done with upside down lids as I was waiting for my SFDs to arrive.





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Edited by urthtown (12/11/13 06:50 PM)


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Offlineurthtown
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Re: slow growth shiitake [Re: urthtown]
    #19264731 - 12/12/13 08:16 AM (10 years, 1 month ago)

Here's the update! Took two transfers - one to a wheat berry petri (actually a ball mason jar) and the other to MEA in a ball mason jar.

Again not concerned if this culture doesn't grow out, this is mostly just to practice technique. What do you guys think of my little culture and where I took my wedges from?



I tried to stay away from what loooks like something yucky taking shape in the top right.


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Invisibleblindingleaf
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Re: slow growth shiitake [Re: urthtown]
    #19264811 - 12/12/13 08:37 AM (10 years, 1 month ago)

hmmm wish i wasn't at work...id post the pics of the plates I have from grocery store shiitaki.  I did 6 plates from the stem.  5 contam'd and 1 did not.  very slow growth, but I transferred to three new dishes, which are growing well, and the initial dish is almost full!  one of the P2 plates was used to inoculate a BRF jar 5 hours ago.  original BRF recipe, not with sawdust...didnt have any, and no where to go at 4 am to get it.  Hopefully it works, if not I have the other plates to work with.

shiitaki grows a bit slower IME than others, maitaki also even slower.  if its clean, no worries!  if it doesn't work, try again!


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Offlineurthtown
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Re: slow growth shiitake [Re: blindingleaf]
    #19264850 - 12/12/13 08:48 AM (10 years, 1 month ago)

Cool! Sounds like a fun project. I am planning on doing sawdust/aspen shaving blocks with wheat germ and gypsum in some myc bags. If this culture looks viable I'll use it to grow out some grain jars - the reason I did one transfer to a grain petri was to see if it grows out well on that. If so I'll take the transfer plate and nocc up some WBS or wheat berries as spawn for the hardwood sawdust blocks.

Please do post your pics later if you remember, I'd like to see for comparison. I did a 30 second h202 dip on this tissue sample and it went all purple so maybe I did some damage and it's taking time to recover but it's good to know it's a slow colonizer on agar.

Do you just transfer as many times as necessary to get a clean culture going on a plate? thanks!!


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Re: slow growth shiitake [Re: urthtown]
    #19265980 - 12/12/13 02:09 PM (10 years, 1 month ago)

Sounds like you are steaming your no-pour agar jars?  They would definately benefit from having a means of GE, so I would use the grain lids next round, these would also help a bit with the condensation issue by helping to release the steam.  Also you should stack the hot jars to cool, as the top jar will pull the heat from the others and reduce condensation on the bottom most jars.


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Re: slow growth shiitake [Re: urthtown]
    #19265986 - 12/12/13 02:10 PM (10 years, 1 month ago)

i didn't do any bleach or h202 dunk for this...just was really careful in SAB.  maybe thats why 5/6 plates con tamed tho?  I was concerned about slower growth after exposure to chemicals.  when u say grain petri...u mean u used BR/WBS/RYE soak water and added to agar powder?  I've been meaning to try that...I have a jar of MEA from mycosupply that u just add water to and PC, all nutrients included.  maybe i could try some grain soak water instead of tap water next time.
gonna take pics in a bit, I promise they will be posted within 2 hours, I work the overnight shift so this is my 7am right now :shrug:


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Offlineurthtown
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Re: slow growth shiitake [Re: blindingleaf]
    #19266155 - 12/12/13 02:40 PM (10 years, 1 month ago)

Quote:

blindingleaf said:
when u say grain petri...u mean u used BR/WBS/RYE soak water and added to agar powder?




Actually just one of my ball mason jars with a SFD lid with prepared wheat berries (12 hr soak,bring to simmer, steam dry, then put them in steam bath for 4 hours as my PC is arriving tomorrow).

Violet called these little plastic containers with 1/2" of prepared brown rice "grain petris" so I went with it. I figure if it colonizes well I can just G2G the small amount of grain into a quart jar, let that colonize and then spawn to bulk.

No worries friend :crazy2: look forward to seeing them whenever you find time.


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Offlineurthtown
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Re: slow growth shiitake [Re: AbbeyRoad]
    #19266164 - 12/12/13 02:43 PM (10 years, 1 month ago)

Quote:

AbbeyRoad said:
Sounds like you are steaming your no-pour agar jars?  They would definately benefit from having a means of GE, so I would use the grain lids next round, these would also help a bit with the condensation issue by helping to release the steam.  Also you should stack the hot jars to cool, as the top jar will pull the heat from the others and reduce condensation on the bottom most jars.




Great advice! Thanks! I'll put those grain lids to use. Looks like my PC will arrive tomorrow so I'll no longer have to steam sterilize for such long periods of time.


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Invisibleblindingleaf
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Re: slow growth shiitake [Re: blindingleaf]
    #19266306 - 12/12/13 03:11 PM (10 years, 1 month ago)

this pic is the first plate that fresh flesh was transferred to, as u can see its almost fully covered.  there was no h202 or bleach, etc dunk, just standard stem tear in half, scalpel, etc.  shiitaki stems are a BITCH to get a small stem piece off, so stringy.  u might b able to make out the 3 places I transferred from.



and this is plate 2, where one of the transfers ended up.



sorry its not on a petri, I have been using PP5 because they are reusable.

yes, transfer as many times to get clean plate, if plate is clean, stop.  u don't want it to compromise the strength of the growth.  usually a grocery store clone will be an isolate, so u just need to transfer for sterility, not for isolation of a strain.

oh shit dude, as I was typing above, u posted, and I too am doing grain PP5 via violet tek.  not grain petris, but small jars:





im experimenting with different kinds of PP5s


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OfflineAbbeyRoad
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Re: slow growth shiitake [Re: blindingleaf]
    #19267158 - 12/12/13 06:32 PM (10 years, 1 month ago)

Those look too small (to be of value) for grains IMO, unless for testing or making a GLC?? 

I do think PP5 will make a splash in the hobby (if not already)and I have recently run some trials in efficiency using PP5 containers, taking LCs directly to substrate in PP5.  When I say directly that means that I pierce the plastic with a hot needle and use those four needle holes for the GE (covered).  Should know soon, but i would say limiting factor with this approach is appropriate GE in larger containers. Unless I can get totally lazy with PP5, I see no motivating factors to leave my jars and lids that work and have already stood up for years :shrug:


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Offlineurthtown
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Re: slow growth shiitake [Re: AbbeyRoad]
    #19267334 - 12/12/13 07:05 PM (10 years, 1 month ago)

Quote:

oh shit dude, as I was typing above, u posted, and I too am doing grain PP5 via violet tek.  not grain petris, but small jars




Haha brilliant, similar thing I just used mason jars and hard red wheat berries.

Quote:

AbbeyRoad said:
Those look too small (to be of value) for grains IMO, unless for testing or making a GLC?? 

I do think PP5 will make a splash in the hobby (if not already)and I have recently run some trials in efficiency using PP5 containers, taking LCs directly to substrate in PP5.  When I say directly that means that I pierce the plastic with a hot needle and use those four needle holes for the GE (covered).  Should know soon, but i would say limiting factor with this approach is appropriate GE in larger containers. Unless I can get totally lazy with PP5, I see no motivating factors to leave my jars and lids that work and have already stood up for years :shrug:




Abbey, we both were looking to Violet's tek for inspiration - this thread in particular is inspirational and challenges your comment that they look too small to be of value. Scroll down and look at some of the flushes and the quantity of pp5 containers. It just seems like a great option to experiment with as a beginner to grow out small quantities of a culture and even fruit directly from the container.

Here's what mine look like:



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Re: slow growth shiitake [Re: urthtown]
    #19269457 - 12/13/13 06:03 AM (10 years, 1 month ago)

yea the really small one (and the 4 others that are that small) are for testing isolates (I usually use them for agar, as a way to save money on petris).  The larger ones will be fruited when fully colonized with a casing, in a similar attempt to test isolates.  the small one is 4 ounces and larger one maybe 8 ounces.  The way I prepped the BR in the small one is way better though, the larger one the BR is too wet, but myc is still growing...albeit slowly

I really want to try a GLC and the PP5 might be the best way to try it out, as u mentioned.  A big thing with Violet Tek is the Bio efficiency, and the fact that there is no shaking, no spawning, etc, so all of the mycs "power" is held intact for fruiting.  she (violet) used ms on agar or grain petris and fruits them, picking out certain fruits for specific reasons and cloning them to get a good iso.  Its a great idea, especially since if u fruit in vitro, there is a way less chance for con tams to play a role in the cloning process.  I highly suggest reading the thread, I was skeptical at first as well (it in linked in last post by OP).  Im experimenting with it, so all of my quart jars are colonizing next to my grain PP5's.


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Re: slow growth shiitake [Re: blindingleaf]
    #19269465 - 12/13/13 06:06 AM (10 years, 1 month ago)

also...abbeyroad...can u expand on ur idea on the LC efficiency u mentioned...?  Im intrigued... u mean u use the PP5 for an LC or u innoc a large PP5 with an lc to test it, or the PP5 has the desired substrate u want to test (a weird one...lets say dog food) and u innoc with lc to see if it grows.... just wondering cause I love experimenting!


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Re: slow growth shiitake [Re: blindingleaf]
    #19269576 - 12/13/13 07:02 AM (10 years, 1 month ago)

I think I'll try making some mycelium water with the jars with the injection ports. Squirt 10 cc of sterile water in, swish around for a few minutes on a fully colonized, clean agar plate and then suck it back up. I saw this thread and thought it was a cool way to approach it too. Just pour some sterilized grain in your SAB into a clean, colonized mason jar agar plate. Wait for it to colonize and then use a syringe to take liquid tissue cultures to grain jars, agar, BRF cakes, etc. Scroll about halfway down to see InTheBiggun's post.

Seems like the easiest way to make GLC and compatible with my mason jar tek and your pp5 experiments. :thumbup:


Edited by urthtown (12/13/13 07:03 AM)


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Re: slow growth shiitake [Re: blindingleaf]
    #19270624 - 12/13/13 12:47 PM (10 years, 1 month ago)

Quote:

blindingleaf said:
also...abbeyroad...can u expand on ur idea on the LC efficiency u mentioned...?  Im intrigued... u mean u use the PP5 for an LC or u innoc a large PP5 with an lc to test it, or the PP5 has the desired substrate u want to test (a weird one...lets say dog food) and u innoc with lc to see if it grows.... just wondering cause I love experimenting!




I inject final fruiting substrate inside an unmodifed PP5 container with LC (ie no ports other than what my hot needle makes).  If Im going to make a lid, it will be for my glass jars.  I have noted that I will start getting deformed PP5 containers (I use Glad food containers)after 2 rounds in the PC so don't think they have the longevity of glass - perhaps your jars are better.  Also not hating, but Violets teks appear to be widely debated.

Also your steps for GLC will work but think your order is incorrect.  I would start on agar, then transfer to grains, then make GLC.  Ive yet to make a bad GLC syringe this way.

I follow this tek= Grain LC, except I use rye berries and never take the lid off.  Ive stripped and recovered a half full 1\2 pint jar three times, and finally spawned it to bulk successfully (although I wouldnt recommend as a practice).


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Re: slow growth shiitake [Re: AbbeyRoad]
    #19271185 - 12/13/13 03:04 PM (10 years, 1 month ago)

I'm still confused...(just woke up) when u say u inject the substrate...u mean like coir or poo cause u wanna test it?  i have never seen a PP5 that big for a grow, unless u wanna test the quality of a substrate or test the ability of the myc in the lc to colonize it..in which case thats a good idea.  either that or u meant grains?


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Re: slow growth shiitake [Re: blindingleaf]
    #19273973 - 12/14/13 10:01 AM (10 years, 1 month ago)

Quote:

blindingleaf said:
I'm still confused...(just woke up) when u say u inject the substrate...u mean like coir or poo cause u wanna test it?





Exactly..

Quote:


i have never seen a PP5 that big for a grow, unless u wanna test the quality of a substrate or test the ability of the myc in the lc to colonize it..in which case thats a good idea.  Either that or u meant grains?





You got my drift.  I'm trying the 64oz containers this way right now for Enoki.  Im small hobbyist, so this is adequate to me. No its final substrate\no grain.  Like I said Im just playing around with being lazy :shrug: If I can use LC, not build ports, not spawn grains, then that would make thing much easier.  Same concept as direct inoculation of sawdust, but I'm trying to cut the corner of having to make injection + GE ports...


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