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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis] * 1
    #15031842 -

Stropharis said:



I'd use those to inoculate grains as-is.  Put each petri section into its own grain master.
RR


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Re: Isolating On Agar and Sectoring (with pics) [Re: EvilMushroom666]
    #15031876 -

EvilMushroom666 said:
What is the agar mix you are using currently?

The nutrient load in your agar mix can play a role in the visual
appearance of your mycelium. The less nutritious your agar is the more
likely that cube mycelium will exhibit rhizomorphic growth. The higher
the nutrient load the more likely you are to see cottony growth
even from a culture that has shown rhizomorphic mycelium on bulk
substrates or agar in the past.

Quote:
Mycelium will readily change states back and forth to and from various types.  Agar with a weak nutrient load will encourage rhizomorphic growth.
RR



It is the same mix that I was using last time although there could be slight differences and it was fresher, thanks for the input. That is good information.


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Re: Isolating On Agar and Sectoring (with pics) [Re: RogerRabbit]
    #15031908 -

RogerRabbit said:
Stropharis said:



I'd use those to inoculate grains as-is.  Put each petri section into its own grain master.
RR


I could still do that seeing as they are in the fridge. My goal is to find a well performing true monoculture though, for use in a future breeding project. Any input on the latest photos?


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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis]
    #15046318 -

I am not entirely sure what the deal is. Like I said previously the cultures began to spread with questionable mycelium but now its starting to show rhizo on the edges. I will take transfers from each, using one to store and one to fruit out. I will have to leave town for a bit so I will toss em in the jars before I go.

There is a lot of experienced people on here with a wealth of knowledge, let me know what you think about this culture. Thanks.



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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis]
    #15046407 -

That is some weird, wispy, cottony looking mycelium that now surrounds the 2 cultures on the right side of my screen.  My guess, contam. spores were present and riding along with the mycelium, caught up to, then passed the leading edges of those 2 cultures contaminating them. 

To prevent this from happening. I take transfers before the culture reaches 1 inch in diameter.  In the case there are contam. spores present, the probability of the myce. outrunning the contam. spores is highest before the culture reaches said diameter.  I would have taken the transfers when the cultures were the size they were in the original post. 

The culture on the bottom left of my screen presently LOOKS un-contaminated.  I'd transfer off of that culture to just FEW grain master jars and either transfer the other 2, questionable cultures to more agar (to check then possibly clean the myce..) or, hot agar pour over the entire dish, or both. 

:peace:

DI


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Re: Isolating On Agar and Sectoring (with pics) [Re: Doctor_Inoc]
    #15061661 -

All of the 18 transfers I took are clean and healthy with the exception of one.  That is 9 isolates to near isolates with one culture to fruit out and the others (duplicates) for temporary storage. Here is a photo of the section with the contam on it… It was one of the dishes to be used for transfer to grains. I separated the healthy mycelium away from the contam to a new dish…. They will soon all be put in the jars and temp storage plates put in the fridge.


Enjoy the feedback as always.



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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis]
    #15392596 -

Lets see if I can sum up this update.

Unfortunately I had to leave on business twice during the past two months, the first time for nearly an entire month, I left shortly after I made the transfers to grain and put the duplicates in the fridge.

The second time I had to leave was just after I spawned 3 of the 9 jars to coir/poo in trays, I would of liked to have finished the other jars but the trip was on short notice, and it was difficult to decide what would be better for the project. I returned to find the remaining 6 jars over colonized, and three cased trays well over colonized and obviously to the point where cell division was at a near halt.  I cased the trays 50/50+ and tossed some casing in the jars as well. I waited till they were ready and put them in the FC. This wasn’t going so well, not to mentioned the climate changes very quickly this time of year. It was now freezing outside and more difficult to maintain ideal conditions. It all needed to be re-tuned.

The 6 jars are coming along, not pinning yet.

The three cased trays are all pinning now but they are all showing similar problems. Mutant knots and primordial formed all over, they are now starting to develop into pins. There are some healthy ones, but mostly mutants. Interesting, especially considering all three are doing it, it leads me to believe that this is due to environment instead of genetics, unless of course this is common when working with isolates via sectoring on agar? I mean, we aren’t even sure they will fruit in the first place. Can a true monoculture have this much phenotype variation?



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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis]
    #15393944 -

How long has the tray in that pic been in fruiting?


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Re: Isolating On Agar and Sectoring (with pics) [Re: LayinUp]
    #15396109 -

LayinUp said:
How long has the tray in that pic been in fruiting?


About 9 Days.


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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis]
    #15396274 -

A few things...

Isolating from multi-spore. If you can then you're better off isolating from a fully grown fruit. When you isolate from multispores you never know what you're going to get until you put in the work to grow them out.

I don't know how many cultures it takes to get signs of degrading isolates, but that might be what you're seeing in your petri dishes after... 5 transfers if I'm reading this right. The weak mycelium is probably just extra yummy nutrients in the agar.


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Re: Isolating On Agar and Sectoring (with pics) [Re: San]
    #15396332 -

Although this was started from a MS culture, it was shortly after I started these cultures that I discovered it was showing signes of senescence. Apparently not something I isolated away from in this case. Maybe some more experienced folks can chime in with a little knowledge on phenotypes and senescence when using this method.


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Re: Isolating On Agar and Sectoring (with pics) [Re: Stropharis] * 1
    #15438819 -

You shouldnt be seeing senescene, expanding Cubey MS on agar...:shrug:

My guess is, when you made your transfer, you cut a decent sized piece of agar...
This gave the mycelium of the transfer a healthy bit of agar to eat while jumping to your new plate...:shrug:
This might account to your new looking growth...
As evil mentioned roger's post, mycelium can change from Rhizo to non-Rhizo and back for no reasons sometimes...

Try making more transfers, but try to just scrape a tiny bit of mycelium off the top instead of cutting a wedge and transferring it...

I been trolling you tonight, reading a few of your posts...
I'll leave you some :mushroom2: rating's in the AM!


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Re: Isolating On Agar and Sectoring (with pics) [Re: total]
    #15439036 -

I noticed your agar looks different then mine. I use 10 gram light malt and 13 grams of agar for 500ml. What agar mix do you use?


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Re: Isolating On Agar and Sectoring (with pics) [Re: EvilMushroom666]
    #15439400 -

EvilMushroom666 said:
What is the agar mix you are using currently?

The nutrient load in your agar mix can play a role in the visual
appearance of your mycelium. The less nutritious your agar is the more
likely that cube mycelium will exhibit rhizomorphic growth. The higher
the nutrient load the more likely you are to see cottony growth
even from a culture that has shown rhizomorphic mycelium on bulk
substrates or agar in the past.

Quote:
Mycelium will readily change states back and forth to and from various types.  Agar with a weak nutrient load will encourage rhizomorphic growth.
RR



so that giant blob i just threw out wasnt the nastys?:ohwell:

and nice work man:wetself:

Edited by senorcafe (11/29/11 08:51 AM)

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Re: Isolating On Agar and Sectoring (with pics) [Re: PinsWellWithOthers]
    #15440350 -

total said:
You shouldnt be seeing senescene, expanding Cubey MS on agar...:shrug:

My guess is, when you made your transfer, you cut a decent sized piece of agar...
This gave the mycelium of the transfer a healthy bit of agar to eat while jumping to your new plate...:shrug:
This might account to your new looking growth...
As evil mentioned roger's post, mycelium can change from Rhizo to non-Rhizo and back for no reasons sometimes...

Try making more transfers, but try to just scrape a tiny bit of mycelium off the top instead of cutting a wedge and transferring it...



The original transfer was taken from a culture that had been running for several G2G transfers. It was not tell after the jar that I had taken the transfer from fruited retarded that I actually understood what subsense was, this happend while in the process of taking these isolates and all of them fruited poorly with lots of mutations. I am finished with this culture and just cleaned out the fridge recently.

total said:I been trolling you tonight, reading a few of your posts...
I'll leave you some  rating's in the AM!


Thanks for reading. Keep in mind that it wasn't till the past 48 hours or so that I posted randomly in OTD. Not the usual, but good time non the less.

Quote:
PinsWellWithOthers said:
I noticed your agar looks different then mine. I use 10 gram light malt and 13 grams of agar for 500ml. What agar mix do you use?


This was done with a premix Malt Extract, 45g/one liter. This will be changing soon and I will be mixing my own, primarily due to connivance in my particular location.


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