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OfflineTerry M
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Photos of monokaryotic vs. dikaryotic mycelium? * 1
    #15976188 - 03/21/12 12:11 PM (12 years, 10 days ago)

Looking for examples of mono and dikaryotic cultures growing on agar. RR has posted a text description of the distinction, but I really need some photo examples in order to recognize the two. I'm at the moment trying out 3 different stains for nucleus visualization under my microscope. 2 failed me so far, but I have high hopes for Crystal Violet!


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Offlinemikesethnobotany
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15976775 - 03/21/12 02:43 PM (12 years, 10 days ago)

:nothingtoadd:

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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: mikesethnobotany]
    #15977154 - 03/21/12 04:00 PM (12 years, 10 days ago)

An update on the staining situation for nuclei. I tried two handy cultures (H. erinaceus and H. capnoides) to see if there was any strain dependency.

Nuclei visible:
Bandoni Safranin O solution* - NO
Methylene Blue - YES, but couldn't get septa to show, even combined with other stains
Crystal Violet - NO

I just ordered the fixings for Acetic Orcein Stain, which is supposed to stain nuclei.

:frown:

*Bandomy Safranin O solution from:
R. J. Bandoni, "Safranin O as a Rapid Nuclear Stain for Fungi," Mycologia, Vol. 71, No. 4 (Jul. - Aug., 1979), pp. 873-874.


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Offlinemikesethnobotany
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15977322 - 03/21/12 04:32 PM (12 years, 10 days ago)

Does DAPI stain work? Thats what we use to stain nuclei of microvascular endothelial cells

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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15977352 - 03/21/12 04:41 PM (12 years, 10 days ago)

From my own experience it´s impossible to recognize the monokaryotic/dikaryotic condition by visual inspection of mycelia in agar plates. You must look for clamp connections when possible. They are very easy to found in dikaryotic mycelia of basidiomycetes (although not in all of them) using a decent microscope.

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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: mikesethnobotany]
    #15977434 - 03/21/12 04:59 PM (12 years, 10 days ago)

Quote:

mikesethnobotany said:
Does DAPI stain work? Thats what we use to stain nuclei of microvascular endothelial cells



I thought you needed a fluorescent microscope to use this stain. I don't have one. :frown:


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Offlinemikesethnobotany
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15977489 - 03/21/12 05:10 PM (12 years, 10 days ago)

:facepalm3: Sorry!

What you can't just go and buy a lil ole fluorescent microscope ; )

Edited by mikesethnobotany (03/21/12 05:15 PM)

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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: troncotron]
    #15985020 - 03/23/12 09:02 AM (12 years, 8 days ago)

Quote:

troncotron said:
From my own experience it´s impossible to recognize the monokaryotic/dikaryotic condition by visual inspection of mycelia in agar plates. You must look for clamp connections when possible. They are very easy to found in dikaryotic mycelia of basidiomycetes (although not in all of them) using a decent microscope.



Good idea, troncotron. So if I find even a single clamp connection between hyphae (but obviously not just bridging septal walls of the same hypha) I can conclude that the whole culture is dikaryotic?


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InvisibleJavadog
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15985212 - 03/23/12 10:15 AM (12 years, 8 days ago)

:popcorn:

Interesting question Terry.

JD


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Offlinemikesethnobotany
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Javadog]
    #15985807 - 03/23/12 12:56 PM (12 years, 8 days ago)

:takingnotes:

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OfflineJimbolino
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15986324 - 03/23/12 03:17 PM (12 years, 8 days ago)

Quote:

Terry M said:
Quote:

mikesethnobotany said:
Does DAPI stain work? Thats what we use to stain nuclei of microvascular endothelial cells



I thought you needed a fluorescent microscope to use this stain. I don't have one. :frown:




can't you use blacklight for excitation? Wiki says blacklight is between 380–315 nm and dapi needs 358nm

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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15994983 - 03/25/12 04:54 PM (12 years, 6 days ago)

Quote:

Terry M said:
Looking for examples of mono and dikaryotic cultures growing on agar. RR has posted a text description of the distinction, but I really need some photo examples in order to recognize the two. I'm at the moment trying out 3 different stains for nucleus visualization under my microscope. 2 failed me so far, but I have high hopes for Crystal Violet!



Here are a couple shots of a dikariotic monoculture. Mono meaning a single sector rather than monokariotic. 


The culture pictured is a Penis Envy Cubensis Isolate.

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InvisibleinskiM
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: ProfessorPinHead]
    #15995058 - 03/25/12 05:14 PM (12 years, 6 days ago)

Here is a micrograph showing the clamp connections that are present in dikaryotic mycelium, take note that not all dikaryotic colonies are capable of producing fertile fruit bodies!


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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: inski]
    #15995630 - 03/25/12 07:23 PM (12 years, 6 days ago)

Quote:

inski said:
Here is a micrograph showing the clamp connections that are present in dikaryotic mycelium, take note that not all dikaryotic colonies are capable of producing fertile fruit bodies!





Those look to me like simply the clamp connections that bridge the septa of the hyphae, not the clamp connections that bridge different hyphae. If they weren't there, the nuclei would be cut off by the septa. They migrate around the septa via these clamp connections so that they can cluster together during mating.

I've got plenty of those!


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Edited by Terry M (03/25/12 08:39 PM)

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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: ProfessorPinHead]
    #15995819 - 03/25/12 07:57 PM (12 years, 6 days ago)

Quote:

ProfessorPinHead said:
Quote:

Terry M said:
Looking for examples of mono and dikaryotic cultures growing on agar. RR has posted a text description of the distinction, but I really need some photo examples in order to recognize the two. I'm at the moment trying out 3 different stains for nucleus visualization under my microscope. 2 failed me so far, but I have high hopes for Crystal Violet!



Here are a couple shots of a dikariotic monoculture. Mono meaning a single sector rather than monokariotic. 


The culture pictured is a Penis Envy Cubensis Isolate.



Yup. But RR talked a couple of times about a macroscopic difference between mono and dikaryotic cultures.

Here's one:
http://www.shroomery.org/forums/showflat.php/Number/10534235#10534235

There's a better one, but I can't seem to find it at the moment.


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InvisibleinskiM
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15996697 - 03/25/12 10:48 PM (12 years, 6 days ago)

Quote:

Terry M said:
Quote:

inski said:
Here is a micrograph showing the clamp connections that are present in dikaryotic mycelium, take note that not all dikaryotic colonies are capable of producing fertile fruit bodies!





Those look to me like simply the clamp connections that bridge the septa of the hyphae, not the clamp connections that bridge different hyphae. If they weren't there, the nuclei would be cut off by the septa. They migrate around the septa via these clamp connections so that they can cluster together during mating.

I've got plenty of those!




I'm not sure I follow this statement...

Quote:

Those look to me like simply the clamp connections that bridge the septa of the hyphae, not the clamp connections that bridge different hyphae




The first part of your statement pretty much defines Clamp Connections, as far as I know there is only one type of Clamp Connection apart from those formed by Homothallic Fungi and that is those that are present in Dikaryons which only form after mating, fusion and nuclear division, they bridge the septum or segments of Dikaryotic mycelium ensuring a differing set of nuclei in each segment and therefore ensuring genetic variation, here is a quick drawing that I hope will illustrate this!

In (a) we have two Monokaryons coming together, note, Monokaryons have septa and look like Dikaryons without clamps, this is known as "Mating"
(b) shows Fusion or "Cytogamy"
(c) shows Nuclear Division or "Karyokinesis".
(d) shows Clamp Connection formation.
And (e) shows a stable Dikaryon.

My images of Clamp Connections above were found in the Pileipellis of a Psilocybe species although it would not have mattered where I acquired the material from for my microscopy as all parts of the fruit body are made up of Dikaryotic mycelium and therefore have Clamp Connections.


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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: inski]
    #15996861 - 03/25/12 11:36 PM (12 years, 6 days ago)

You are absolutely right, inski. I was just reading in more detail about clamp connection formation. I was just about to post that I think that I'm full of shit, when I saw your new post! :laugh:

The hyphae don't need clamp connections when they are monokaryotic. The nuclei don't need to bypass the septa to sort themselves into pair, like they do when becoming dikaryotic.

This is great news for me, because now I can easily determine mono vs. dikaryotic mycelium by just looking at them at 1000X, oil immersion. I've got some old cultures which may be monospore. My records were really bad on these, and I'm just not sure. Can't wait to look at the suspected monokaryotic cultures tomorrow.


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InvisibleinskiM
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15997078 - 03/26/12 12:41 AM (12 years, 6 days ago)

Good luck and also, you don't really need to use the 100x oil immersion objective to see clamp connections, I use the 40x objective and see them just fine at 400x, I have been looking at different cultures for quite some time now to determine the presence of Monokaryotic and Dikaryotic colonies and have recently looked at some that I suspected were contaminated, the problem was that the multi spore cultures were too young and had not had time to mate and form stable Dikaryons yet, I trashed two dishes because of this mistake:sad:

It was a rare species also, luckily I have isolated a few good cultures now and my experimentation can continue, my new laminar flow hood has made all of this work so much easier, whenever I do transfers now I cut a small wedge for microscopic examination!

What characteristics make you think you have Monokaryotic mycelium, and what genus and species are you referring to?


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OfflineTerry M
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: inski]
    #15997584 - 03/26/12 06:42 AM (12 years, 5 days ago)

Quote:

inski said:
Good luck and also, you don't really need to use the 100x oil immersion objective to see clamp connections, I use the 40x objective and see them just fine at 400x, I have been looking at different cultures for quite some time now to determine the presence of Monokaryotic and Dikaryotic colonies and have recently looked at some that I suspected were contaminated, the problem was that the multi spore cultures were too young and had not had time to mate and form stable Dikaryons yet, I trashed two dishes because of this mistake:sad:

It was a rare species also, luckily I have isolated a few good cultures now and my experimentation can continue, my new laminar flow hood has made all of this work so much easier, whenever I do transfers now I cut a small wedge for microscopic examination!

What characteristics make you think you have Monokaryotic mycelium, and what genus and species are you referring to?




Again, thanks for correcting me. And that's a very important thing to know about multispore cultures not having the chance yet to mate.

The reason I'm suspecting I may have some monokaryotic cultures is that in order to isolate some very contaminated wild spore prints, I made a series of dilutions of a syringes containing the spores -- 10X, 100X, 1000X, etc. The idea was to get spore mycelium on agar plates that were now separated enough from their contaminates so I could cut them out and culture them on their own plates.

This worked, but now I'm afraid that I've got single spore colonies. I may have even mistakenly taken some samples from the same monospore colony and saved only these. Of course the later could never breed. And if I have too few different monospore colonies, I may not have enough compatible mating types to yield good, fertile strains.


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OfflineProfessorPinHead
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15997664 - 03/26/12 07:39 AM (12 years, 5 days ago)

I'm post rating this thread. :thumbup:

This is some good morning reading, :coffee:

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InvisibleinskiM
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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15998654 - 03/26/12 12:44 PM (12 years, 5 days ago)

All you can do now is experiment with your suspected Monokaryons by inoculating petri dishes with different combinations of the saved cultures to observe whether or not mating will occur, remember, if you are working with a tetrapolar(four spored) fungus each Monokaryon is compatible with only one quarter of it's siblings so for example, if you have 100 Monokaryotic cultures it is possible to create only 25 viable Dikaryotic cultures.


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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #15999378 - 03/26/12 03:20 PM (12 years, 5 days ago)

Quote:

Terry M said:
Good idea, troncotron. So if I find even a single clamp connection between hyphae (but obviously not just bridging septal walls of the same hypha) I can conclude that the whole culture is dikaryotic?





If the cultures are full of clamp connections you can be sure that it´s dikaryotic. Nevertheless nuclear migration takes some time, although it seems to be relatively quick (mm per hour). Sometimes it´s possible to find monokaryotic zones in dikaryotic cultures.
Very nice pics inski:thumbup:
Here are some pics of an Agaricus blazeii culture lacking clamp connections, which are rarely found in this species.


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Re: Photos of monokaryotic vs. dikaryotic mycelium? [Re: Terry M]
    #20800693 - 11/06/14 10:03 AM (9 years, 4 months ago)

Hi Terry,

Could you finally find out how to find differences between mono and dikaryotic mycelium in agar plates? Because I am doing some crosses in the lab with the fungus that I am working on and I can clearly see clamps but, I want to isolate the "new dikaryon" in another plate but I don't think I will still continue seeing clamps.... will I?

Pleeeease tell me you know more about this

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