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Zwinst
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microscoping mycelium on agar plates without opening them - possible?
#28412249 - 07/29/23 01:54 AM (9 months, 15 days ago) |
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Hi, i finally want to get started creating monokaryons for crossing. I realise i have to check the final mycelium for clamp connections and that i need at least 300-400x magnification to do it. Problem is, i would have to remove some mycelium from the plate, put it on a slide and then put it under my microscope. Which costs a lot of time and effort and creates opportunity for contamination. With the 10x lens (125x) i can put the whole plate in, but thats not enough magnification. With the 40x lens (500x) the working distance is too short to get anything in focus.
Are there affordable microscopes or lenses that enable me to just use the whole plate?
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VP123
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst] 1
#28412360 - 07/29/23 05:00 AM (9 months, 15 days ago) |
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There are long working distance objectives. I have never used them but they exist. They are not cheap as far as I know.
I assume you are using an upright microscope rather than an inverted microscope. You could try flipping the petri dish in the microscope stage and see if you can get a clear enough image through the agar layer. Depending on your agar it may be enough for your purposes. Or you may have to make thinner and more transparent agar layers. I just hope this works for you.
Edited by VP123 (07/29/23 05:01 AM)
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Zwinst
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: VP123]
#28412438 - 07/29/23 06:42 AM (9 months, 15 days ago) |
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I already tried flipping them around. The distance is still way too big. Pouring thinner isn´t going to help either, i have to get the hyphae within less than a milimeter in order to focus on them. Yes, i have an old standard upright microscope (with 70s leica lenses and newer zeiss eyepieces) and i know of those expensive long working distance objectives. The cheapest ones i found were still 400 bucks though... thats not happening anytime soon.
I was just wondering if anyone has found a workaround yet or something that is affordable.
I have also seen those cheapo chinese ccd microscopes with lcd screens. They claim between 400x and 600x depending on the model and a working distance of >15mm, which would be perfect. Has anyone experience with those? If they are good enough to see the clamp connections, i would buy one. But somehow i doubt that very much.
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trippleblack
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst]
#28413187 - 07/29/23 08:02 PM (9 months, 15 days ago) |
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I purchased long working distance lenses to properly work under my am scope. they where moderately expensive but well worth the money.
i have never had a big problem flipping thin plates and analyzing 400x, you see a mono mark that spot with a marker on the petri, extract that agar section, and hope that it remains a mono. Thin pours are best.
My favorite method is just removing the cover off the petri dish and lowering the microscope all while in front of the flow hood -this has never led to infection -i would just wipe the scope down, and place it in front of flow hood for a couple hours before use.
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covertjoy

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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst] 2
#28413360 - 07/29/23 11:04 PM (9 months, 14 days ago) |
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I'm still learning but so far I've had the most success with preparing a slide after doing a transfer and viewing with darkfield 400x. So I do my transfer and then create the slide from what remains in the original dish after the transfer is complete. I take a small square of agar/mycelium about 1-2mm square, put it on a slide and then press a slide cover onto it to flatten it out.

Excuse the poor quality image, the camera that came with my second hand microscope leaves a lot to be desired.
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: covertjoy] 1
#28438116 - 08/18/23 11:48 AM (8 months, 26 days ago) |
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Bismillah
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst]
#28707972 - 03/22/24 02:17 AM (1 month, 26 days ago) |
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Did you find a solution that works? I'm very much a noob at this, although I had microscope classes in uni 20+ years ago I picked up a 40x with a corrective collar for 0-2mm cover slips. It works fairly well when I pour water agar very thin. In other plates I've cut out a "window" in the agar for the myc to grow freely on the plate bottom. Here I get much better resolution. Not as good as a mounted specimen though, which is likely due to the distance to the condenser lens is far from optimal.
Another thing I've found is that it's possible to find clamps at 100x. The 10x objective is much more forgiving to the light source, and it's nice to have such a wide field of view. I've been going back and forth with monos and dikaryons, and I think it's just a matter of practice to reliably rule out clamps at 100x.
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Zwinst
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Bismillah]
#28708599 - 03/22/24 01:42 PM (1 month, 26 days ago) |
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No, i haven´t found a solution. But since my hepa is working great, i am just sticking to the old transfer method...
You can see clamps at 100x?! 
I am struggling to reliably identify them at 500x. Inadequate sample preparation may play a role though.
Edited by Zwinst (03/22/24 01:44 PM)
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Bismillah
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst] 1
#28708769 - 03/22/24 04:19 PM (1 month, 26 days ago) |
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I can see the lack of clamps at least. For suspected clamps the 40x helps a lot.
Like this one at 100x, I wasn't sure about the bumps in the middle. At 400x I'm pretty sure they're not clamps. This is taken through the bottom of the dish, agar removed for better viewing. Iphone on eyepiece, so it's sharper in real life.
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Zwinst
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Bismillah]
#28708969 - 03/22/24 06:50 PM (1 month, 26 days ago) |
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There seems to be a fundamental lack of understanding the problem here. What you present in your picture could very well be dicaryotic mycelium, since at this magnification you CAN NOT see clamps. Auf Deutsch: Leg dein Eifon weg und guck bei 500x durchs Okular. Das Bild sieht ja echt nett aus, hat aber keinerlei Wert. Look at this. Thats a clamp connection.
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Bismillah
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst]
#28709803 - 03/23/24 11:01 AM (1 month, 25 days ago) |
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I can see clamps fine at 100x. Yes I will probably miss a few, and some are hard to separate from a budding branch, but I can see them. I can post some pics at 400x and 100x later so you can see what I mean, in the mountains atm.
The theoretical resolution of a 10x objective is around 1 micron, so there's nothing far fetched about this.
Edit: I found this pic from a month back. This is a dyed mount at 1000x.
 Switching back between 400x and 100x, I realize the bumps I saw at 100x were clamps. I've eliminated a few dikariotic cultures just using the 100x. For suspected monos I'll still go back and check and recheck. I will probably also be making slide mounts, since 400x through the petri gives pretty poor contrast and resolution..
There's is also a chance that I'm completely wrong. I've only been at this for a couple of months.
Edited by Bismillah (03/24/24 03:43 AM)
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Bismillah
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Bismillah] 1
#28721120 - 03/31/24 08:46 AM (1 month, 17 days ago) |
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I did some testing on a dish that's going out anyway. So this is dikaryon pan mycelium on water agar. I can get a pretty good 100x view from the bottom of the dish, especially where I've cut out some agar so the myc grows in air. The refractive index of mycelium and air is very different, thus I get better contrast in brightfield than if I mount the slide.
Here's the dish, with mycelium grown out on the cutouts:

Here's using a 10x achromat (0.22NA) from the bottom:

Note that this is taken with the wide angle lens of my iphone (I don't have the fancier 3-lens version with a "normal" lens). The app (halide) is set to upsample 2x to crop out the image circle. On the right is a manually cropped and upscaled image that I've tried to keep at a fixed magnification.
Here's using a 40x plan achromat (0.55NA long working distance):

The resolution is better, although worse than expected from a 40x since the condenser is so far away from the sample. I see no change in the image when I stop down the condenser to 0.2NA.
The agar is pretty hard, so a squish mount was almost impossible. The agar just slipped out under the cover slide. I used transparent tape instead to grab some myc from the "window" in the dish, and stuck it to a cover slide. As before, the "full" image is on the left, while I tried to keep the image on the right at about the same magnification.
10x objective:

40x objective:

100x objective:

Ok, so I didn't spend any time tweaking settings for better images or post-processing for that matter: I work with binoculars, and the view I get is a lot better than this. Anyway, to conclude:
- 100x through the bottom of a dish serves a useful purpose to identify dikaryons.
- 400x using a LWD objective gives a bit higher confidence if unsure.
- 1000x is nice if you want to see the pimples on the clamp.
Edited by Bismillah (03/31/24 10:39 AM)
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idimos
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Zwinst]
#28766009 - 05/08/24 03:35 AM (10 days, 18 hours ago) |
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I don't think you will find a practical and affordable way to achieve that. Physics sucks
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LogicaL Chaos
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: Bismillah]
#28773025 - 05/13/24 01:01 PM (5 days, 8 hours ago) |
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Looks like you figured it out!
Seems like the best way to do this would be a thin, glass petri dish and use really clear agar.
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Re: microscoping mycelium on agar plates without opening them - possible? [Re: LogicaL Chaos] 1
#28773764 - 05/14/24 04:35 AM (4 days, 17 hours ago) |
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I use plastic petri dishes and pour the agar really thin. Also using water agar or low nute agar (I use 0.3%) helps keeping the mycelium thin. I've found cutouts aren't really necessary, only if you get dense growth on high nute agar.
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