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Invisiblemetacohl
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Re: Lepista nuda [Re: Javadog]
    #15827939 - 02/18/12 03:50 PM (8 years, 8 months ago)

Quote:

Javadog said:
Two days on Javadogs own WPDWYA* agar and everything looks better so far:


I was hoping to make my PMEA tonight, but I am being dragged to
HELL^H^H^H^H the in-laws today.  Ah well.

Take care,

JD

* Wrong Potato Dextrose Wrong Yeast Agar  ;0)




Looking nice JD! And good luck with the in-laws... :wink:

Carsten: I was not aware of that starch test using iodine. Here is what I found about the reaction.

"What properties of starch (given its chemical structure) allow it to be used as an indicator?
Davender Khera, Yale University
When starch is mixed with iodine in water, an intensely colored starch/iodine complex is formed. Many of the details of the reaction are still unknown. But it seems that the iodine (in the form of I5- ions) gets stuck in the coils of beta amylose molecules (beta amylose is a soluble starch). The starch forces the iodine atoms into a linear arrangement in the central groove of the amylose coil. There is some transfer of charge between the starch and the iodine. That changes the way electrons are confined, and so, changes spacing of the energy levels. The iodine/starch complex has energy level spacings that are just so for absorbing visible light- giving the complex its intense blue color."

Pretty interesting. I don't think that the similarity in color produced by the reaction of L. Nuda enzymes with the starches necessarily means a similar mechanism, but perhaps.

Also, all of my tissue cultures and subcultures from my soil sample are pretty much annihilated by bacteria. All of my tissue cultures pretty much dissolved into a pool of bacterial sludge :frown: Did you guys find it easy to germinate the spores on agar? Any info on how common fruiting isolates are?


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Edited by metacohl (02/18/12 04:12 PM)


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InvisibleJavadog
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Re: Lepista nuda [Re: metacohl]
    #15830563 - 02/19/12 04:10 AM (8 years, 8 months ago)

I survived.

...and I poured a sleeve of PMEA dishes before I left.

First L. nuda xfers onto it:


I will let you know how it grows out.

JD


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Re: Lepista nuda [Re: Javadog]
    #15830699 - 02/19/12 05:40 AM (8 years, 8 months ago)

@JD
Really nice looking Plates! Your WPDWYA seems not to be W at all. Cool that one can see the metabolic activity without even clicking on the thumbnail. Can't wait to see all the new plates in two weeks.

@metacohl
All we can say for sure is that it is some kind of two component reaction, where something from the mycelium interferes with something in the agar medium. My starch theory is just a possibility, based on colors and the known preference of L. nuda for starch. The problematic part is no staining on corn steep agar, which would mean there is either no starch or some type which reacts without color. Both may be unrealistic.
I found several LC recipes, calling for 4% starch, but I wonder where starch would occur in leaf or needle litter in nature.
And yes, germinating L. nuda spores is easy, though it may take two or three weeks.

Carsten


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Invisiblemetacohl
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Re: Lepista nuda [Re: Mycelio]
    #15831435 - 02/19/12 11:48 AM (8 years, 8 months ago)

Quote:

Mycelio said:
@JD
Really nice looking Plates! Your WPDWYA seems not to be W at all. Cool that one can see the metabolic activity without even clicking on the thumbnail. Can't wait to see all the new plates in two weeks.




Right! Such a great color too! I'm very interested to see how the PMEA plates turn out as well.

Quote:

Mycelio said:
@metacohl
All we can say for sure is that it is some kind of two component reaction, where something from the mycelium interferes with something in the agar medium. My starch theory is just a possibility, based on colors and the known preference of L. nuda for starch. The problematic part is no staining on corn steep agar, which would mean there is either no starch or some type which reacts without color. Both may be unrealistic.
I found several LC recipes, calling for 4% starch, but I wonder where starch would occur in leaf or needle litter in nature.
And yes, germinating L. nuda spores is easy, though it may take two or three weeks.

Carsten




Thanks for the input, glad to know the timeframe for germination. Everything you have said about the color reaction makes sense. I also wonder where in a pile of leaf or needle litter L. Nuda would find starch...

After thinking about your comment of no visible color change on corn steep, I decided to do look into the starch content of potatoes vs. corn.

http://members.home.nl/ajansma/zetmeel/infoe/chapter2.htm
"The ratio amylose/amylopectine differs per starch source. Table 2.3 indicates the composition for a number of starches.

starch amylose amylopectine
potato 21 79
maize 28 72
wheat 26 74
tapioca 17 83
waxy maize - 100"

It seems all starch is a mixture of amylose and amylopectine, just at varying ratios?


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Re: Lepista nuda [Re: metacohl] * 1
    #15831641 - 02/19/12 12:42 PM (8 years, 8 months ago)

Thanks guys. 

(this thread started to rock when you arrived Carsten)

FYI, I went ahead and purchased a lb. of the corn steep, just to have
another agar alternative.

This all kinda cracks me up, as with most species it has been the
fruiting stage that has been the hard part so far.  All of this
fighting with agar and grains rather takes me back to my early
struggles.

Take care,

JD


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Re: Lepista nuda [Re: Javadog]
    #15837520 - 02/20/12 03:02 PM (8 years, 7 months ago)

I'm doing some experiments with Lepista nuda, too. I obtained my culture from a wild specimen found on mostly spruce needles humus.



In my case, mycelium grows VERY slowly on PDA (can't complete an entire plate in 2 months), but with a very strong purple color. On MEYA it grow much faster, but very aerial and with very pale purple shades, like this:



I prepared a spawn jar (mostly millet) and inoculated with an agar wedge; after over a month, it fully colonized with aerial, apparently weak mycelium. On 06th of January I spawned the jar on a 2,5 kg sawdust bag, in order to obtain more spawn. It colonized it, without liking it very much but sufficiently strong to keep sawdust clumped. Pic was taken on 18th of January. Now bag is fully colonized)



Now I'm planning to spawn it on pasteurized cow manure/straw, and see what'll happen :grin:


--------------------


Edited by atricoz (02/20/12 03:02 PM)


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Re: Lepista nuda [Re: atricoz]
    #15837758 - 02/20/12 03:50 PM (8 years, 7 months ago)

Thank you for sharing Atricoz. 

It is very interesting, just how different your growth is from what I am seeing.

For my part, I have to report that I might be seeing bacterial colonies
on my PMEA dishes.  If they do develop, then I will make another sleeve
of them, but will heat the mix longer on the stove, and in the PC.

The WPDWYA is still clean and growing without the blackening die-back
that I am working to prevent.

Onward and upward,

JD

P.S. I thought to add that this is a situation where I could have used
glass petri dishes.  I love the disposables, but begin to think that
I will benefit from a set of glass dishes.  This would allow me to
re-sterilize dishes when I am experimenting with new agar recipes.


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Edited by Javadog (02/20/12 03:54 PM)


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Re: Lepista nuda [Re: Javadog]
    #15849935 - 02/22/12 11:49 PM (8 years, 7 months ago)

Hello all,

I have a few things to add.

While the jury is still out, the WPDWYA dishes do seem to be staving
off the mycelial die-back that plain and antibiotic agar suffered from.

Here is a photo comparison:


(sorry, the flash really accents the fogging)

This evening I made up six jars of WBS and mixed it with some dry compost.
The result was on the dry side, as Carsten suggested.  I will be
inoculating these jars with the WPDWYA agar dishes.

We will see if anything comes of it.

Also, I went ahead and invested in a dozen pyrex petris.  I have a huge box
of sleeves of the disposable variety to use more commonly, but I wanted
a set of the glass variety with which to experiment.

I am going to resterilize the PMEA dishes.  I want to see if they can be saved.

Take care,

JD


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Re: Lepista nuda [Re: Javadog]
    #15850081 - 02/23/12 12:17 AM (8 years, 7 months ago)

Quote:

Javadog said:


Here is a photo comparison:



JD




You look like your growing contamination.

Lipa


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Re: Lepista nuda [Re: lipa]
    #15850145 - 02/23/12 12:34 AM (8 years, 7 months ago)

Really?!?  Dude, I know zip about this species.

What sort of contam?

I do know that I started off on MEA and took just the leading edge
when I transferred to an anti-biotics MEA dish, and then I did the
same to this PDYA.

What else to do?  A hot pour?

I have seen others grows of this species (i.e. the German website
that Carsten pointed to) and those other efforts also had a large
amount of coloration.

I am open to any suggestion.  I also have a spore-print from the original fruit.

Any ideas appreciated.

Thanks for taking the time,

JD


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Re: Lepista nuda [Re: Javadog]
    #15850195 - 02/23/12 12:44 AM (8 years, 7 months ago)

Sorry to see the difficulties JD, keep on keepin' on!


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Re: Lepista nuda [Re: Javadog]
    #15850392 - 02/23/12 01:43 AM (8 years, 7 months ago)

None of my cultures have ever done that from any blewits in our area. Just saying. I've seen violet tints in the mycelium but no strong coloration like that.

Lipa


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Re: Lepista nuda [Re: lipa]
    #15850402 - 02/23/12 01:46 AM (8 years, 7 months ago)

Well, well. 

I did get an ID from the ID thread....

Here is the fruit:


I will plow forward.  I can only hope that I am blazing trails. (no pun intended ;0)

JD


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Re: Lepista nuda [Re: Javadog]
    #15850419 - 02/23/12 01:50 AM (8 years, 7 months ago)

I could be wrong. It was only a suggestion. I just haven't seen this before.

Lipa


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Re: Lepista nuda [Re: lipa]
    #15850480 - 02/23/12 02:16 AM (8 years, 7 months ago)

No worries.  I will post results, if any.

JD


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Re: Lepista nuda [Re: Javadog]
    #15860996 - 02/25/12 10:40 AM (8 years, 7 months ago)

Well after about ten days I began to see something on the plate I inoculated with spores from my suspected L. Nuda specimen. Not much has happened in the four days since then. There appears to be a very faint, practically invisible, but coarse, fiborous mycelium growing across the plate. There are multiple germination sites and obvious differences in morphology.

The whole thing is growing SOOOOO SLOW! Just in the past day or so some barely visible white wisps have begun to appear which seem like the beginning of more cottony hyphae. I wish that I could take a picture, but I really don't think my camera could capture the image. At the rate it is growing it could be quite some time before a picture is feasible.

Im just wondering if this description sounds similar to what those of you who have germinated spores of this sp. observed......


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Re: Lepista nuda [Re: metacohl]
    #15862081 - 02/25/12 04:27 PM (8 years, 7 months ago)

Hey metacohl,

Remind me of the agar recipe that you are using...are you seeing any
discoloration of the agar as it is metabolized?  Photos?

For my part, I recall fairly aggressive growth.

I took some new photos of the comparison of growth on the two different agars.

I will dig up my log and confirm the recipes and dates, but I they are
MEA and PDYA (WPDWYA ;0):



The growth appears faster on PDYA, and there does seem to be less of
the blackening die-off, or whatever it is, but there is some on the
PDYA as well.

I went ahead and inoculated six quarts of WBS that I prepared by adding
dry compost (extremely aged manure really) to the grains immediately
after they were boiled and then strained.  I let the compost take up
the extra moisture.  They result was a relatively dry mixture.

The dryness is one factor that I will have to learn by screwing up,
but the result has not suppressed growth, as I am already seeing some action.

The PMEA dishes are very interesting.  Not "successful" at all but interesting.
Actually, I checked as I write this, and the does appear to be growth
jumping off the transfer finally.  We will see if it stays vigorous,
but it is certainly slower.

I reported earlier that the PMEA dishes may have been showing bacterial
colonies (wet, colorul spots) but this may not be the case.  It may
just be how the PMEA forms as it cools.  I begin to wonder this because
I have never seen the spots grow.  I just noticed them initially.

Onward and upward,

JD


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Re: Lepista nuda [Re: Javadog]
    #15862476 - 02/25/12 06:16 PM (8 years, 7 months ago)

Its on PDYA, no discoloration so far.... honestly... I am thinking mold at this point.

Here is the best pic I could get. The faint circle is the border of the almost invisible mycelium I spoke of. It looks fairly coarse and fibrous as I said, but now it is getting this little while dots all over it... I have been trying SOOO hard to get a good picture of this plate today. I am having a lot of trouble with reflection off the top of the dish, but I have no room without an overhead light source.



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Re: Lepista nuda [Re: metacohl]
    #15866826 - 02/26/12 06:37 PM (8 years, 7 months ago)

Uhm, It looks like mold to me :crazy:
Just wait some more before tossing it to see what'll happen. For what I can see, L.nuda cultures are very variable


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Re: Lepista nuda [Re: atricoz]
    #15866890 - 02/26/12 06:52 PM (8 years, 7 months ago)

I know I don't have to be too optimistic with this project, because this isn't a very simple species to grow and that's my first try with it. But this is really exciting :smile:
I spawned the sawdust block in an about equal quantity of pasteurized cow manure, with some pine needles and dry leaves. I made a sort of monotub spreading layers of sawdust and compost. After just one day, mycelium shows signs of awakening. The violet color is just amazing :smile:









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