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Acid-Fast


Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis
#23565580 - 08/22/16 10:44 AM (7 years, 5 months ago) |
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I noticed that the macroscopic morphology of one of my P. Cubensis isolates looked different from the rest of the set of agar plates from the same syringe, even possibly mixed with mold, so I did a cellophane tape mount with lactophenol aniline blue and saw only septated hyphae, no spores (conidia/ascopores) were present. This is good.
Is there any good literature/micrographs about/on stained tissue sections of P. Cubensis. My go-to-source for microbiological literature is NIH and, alas, P. cubensis is not a pathogen.
Colonizing mycelia isn't interesting to look at, I was just ruling out contamination, but I'm sure other sections of structures might be (i.e. fruiting bodies?)????? I'm really interested in the dynamics of the microscopic morph and how events progress and structures change.
I'm hoping some mushroom geek/guru has authored a fine book on this stuff with real micrographs, not some drawn picture.
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Acid-Fast


Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23567045 - 08/22/16 07:18 PM (7 years, 5 months ago) |
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Ethno-mycology/biology related writings with some micrographs, some interesting stuff. Best information literature I can find so far. I guess I should spend some cash on a triocular scope or binocular attachment to make my future posts in this forum more interesting?
UTFSE, huh? Looks like Workman "Psilocybe Microscopist" has a lot of references and pics I'm looking for. Thanks to him and his posts I can find something interesting to read and see.
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Acid-Fast


Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23645653 - 09/14/16 07:43 PM (7 years, 4 months ago) |
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Okay folks, I shelled out some cash on a scope and camera so this will not only serve as a review of the scope but also a continuation on the topic of cubensis microscopy and employing good quality control measures for cultures.
This is the scope I bought and a link to the Amazon listing. https://www.amazon.com/AmScope-B120C-E1-Siedentopf-Magnification-Illumination/dp/B009VUPIKM/ref=sr_1_1?ie=UTF8&qid=1473902423&sr=8-1&keywords=b120c-e1[/url] So far, I'm not very impressed with LED lighting because it scatters way too much. Granted, I don't have a Nikon or other top of the line LED scope but still, it's white/blue light that scatters too much. Also, it should be mentioned here that it is a 1.3MP camera.
Here is a pick of an ATCC E. coli Q.C. slide...
 Reds do not show up very well at all on this LED scope. Reds are distinctly red on a halogen scope.
Here is a pick of an ATCC S. aurues Q.C. slide...
 Blues come out very blue and they seem have better resolution and even less scattering around the outline of the cells as compared to gram-negative organisms.
Now on to a pick of a scotch tape prep with methylene blue. The supplies...

The suspicious plate in question has a brown to black reverse, none of my other plates had this. Mind you this has also been refrigerated and is growing on SAB. I experimented with SAB, IMA and MEA because it's been many years since my last grow and I wanted to experiment with different medias and the morphologies they induced. It could be just some metabolite/pigment diffusion but here have a look see.

As you can see it looks a little different from my awesome isolate here below. BTW, I found there was a specific technique to properly cloning rhizomorphic growth.

And now for my the scotch tape preps 100x...

and 400x...

I think all looks pretty normal but I haven't stained any of my "normal" isolates yet. What do you think? I'll get around to staining/viewing the others for comparison here in about a day. It's been a long day, working some long hours.
Edited by Acid-Fast (09/14/16 07:49 PM)
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Acid-Fast


Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23646692 - 09/15/16 06:49 AM (7 years, 4 months ago) |
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Pulled another plate from the fridge. This one has a normal white-ish reverse.
100x...

400x...

Odd that I can't find any of what appeared to be nuclei in the brown-black reverse plate. I'm gonna see if the brown-black reverse morphology matches up with any micrographs in the lab today.
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Acid-Fast


Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23648559 - 09/15/16 07:31 PM (7 years, 4 months ago) |
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Let's see how this scope holds up to viewing through agar as this is the most important feature I'm looking for.
The supplies: cornmeal agar tween 80 or any other transparent media with a large cover slip. We allow this to grow under the coverslip so as not to disturb the layers by placing afterwards.

ATCC Cryptococcus neoformans on cornmeal agar tween 80 100x...

400x...

In summary, this scope will work well for my purposes. I wouldn't recommend it to anyone who needs good micrographs but I'm happy that it works with agar because if it didn't I would've returned it. It's got a really tight clearance on viewing plates so I would recommend the slims for this model but it works well enough with the standard plastic plates.
I'm gonna post some pics using this method from spore under a different thread, once I get some more spores.
Edited by Acid-Fast (09/15/16 08:11 PM)
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Greg
always learning




Registered: 10/28/15
Posts: 1,536
Loc: an autoclave
Last seen: 3 months, 3 days
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23649987 - 09/16/16 09:29 AM (7 years, 4 months ago) |
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Regarding your question in my thread over at the microscopy forum, I think your USB camera's white balance is set incorrectly. Not sure how you change it though, I use a totally different camera setup.
Here is a recent picture for example, it's very high resolution compared to that USB camera:
 This picture is completely unedited.
This model of scope is actually quite good IMO, that usb camera just isn't.
Here is another picture, this one is Saccharomyces cerevisiae Lalvin K1-V1116 wine yeast. I couldn't get all the yeast on one plane so it's a bit blurry in places.

The images in that other thread were taken without the camera adapter that I now use. I just positioned the camera lens above the eyepiece which resulted in some very low quality and poor contrast photos.
Now that I have the camera adapter I don't generally need to edit the pictures anymore.
Edited by Greg (09/16/16 09:52 AM)
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Acid-Fast



Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Greg]
#23650969 - 09/16/16 05:00 PM (7 years, 4 months ago) |
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Thanks for the reply Greg. I realized I was in the wrong forum when I found the microscopy forum tucked under the mushroom hunting and ID category. This forum is definitely delegated to research/advanced discussions on things like cross-genera or cross-species mating and what-not. Things I have no experience in. I'm glad I found the forum where I need to be.
I think I'm also learning better how to control the combination of the condenser distance from the stage, the aperture size of the diaphragm, and the intensity of the light with LED. You have to really narrow in on your settings with an LED whereas with a good halogen it's pretty simple to calibrate.
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Greg
always learning




Registered: 10/28/15
Posts: 1,536
Loc: an autoclave
Last seen: 3 months, 3 days
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Acid-Fast]
#23650982 - 09/16/16 05:05 PM (7 years, 4 months ago) |
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The condenser setting definitely makes a huge difference when it's dialed in properly. The aperture hasn't seemed to make much of a difference as long as it covers the entire FOV.
Have you tried the oil objective yet? Super low DOF so it's hard to focus sometimes, especially if your specimen isn't all on the same plane.
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Acid-Fast



Registered: 07/31/16
Posts: 24
Loc: The Hood (Laminar Flow)
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Re: Microscopic morphologies (LPAB/MB) and tissue sections of P. cubensis [Re: Greg]
#23652031 - 09/17/16 02:22 AM (7 years, 4 months ago) |
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Greg -
That's what the pics of E. coli and S. aureus are - 1000x. Controlling the size of the aperture is best used on wet mounts and agar. Going all the way in to 2500x (25x ocular lenses and the 100x objective) is really unnecessary, IMO. I think such magnification is pushing on the limits of optics. You can only go in so far before you start hitting on the limit ,like wavelengths, and stuff doesn't become coherent. For instance 1 micron = 1000 nanometers. The size of a bacterium like E. coli = ~2 microns and blue light, for instance, = 475 nanometers.
Now that you know we can view stuff on transparent solid media let's transfer some wedges or spores onto some agar. If transferring wedges, let it grow for maybe 2-3 days so we know where to place the sterile coverslip and then let it grow under the slip. Or just start from spore. If we make several plates then we won't have to worry about creating a sterile field over our scopes. Let's get this party started. 
Edit: but if you're looking for time lapse, you'll need a sterile/aseptic field. Would have to clean everything down under a hood or in a box and maybe use a clean turkey bag to cover our objectives, stage, condenser and light source. Be careful not to dirty your optics with any liquids
Let's continue our discussion under microscopy. I'd even appreciate it if a mod could move this thread to microscopy cause that's where it goes. Thanks.
Edited by Acid-Fast (09/17/16 02:30 AM)
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