First Stain, and mounting

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NK7Z
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First Stain, and mounting

#1 Post by NK7Z » Mon Jun 10, 2019 2:45 am

Hello,
Just did my first stain and mount. I carefully dissected an onion, removing a very thin peel of skin from the inside of the onion. Got it laid out on the slide, placed a small drop of water on it, then carefully put the cover slip on, by lowering it, starting at a 45 degree angle, and using a needle to lower it the rest of the way. Once finished, I added a drop of stain to one side of the cover slip, used a paper towel to suck the stain under and across the cover slip, such that the stain surrounded the onion skin.

I then did some cleanup on the slide, and let the stain soak while I cleaned up the dissecting station I built. Took about five minutes... Here is what I noticed:

1. Cover slip was being held above the slide surface by the onion skin, such that it could rock a bit. Is this normal?
2. The stain did not penetrate the onion skin very deeply. Just around the edges.
3. Many intact cells were clearly visible, and with some light diaphragm adjustment, the nucleus of the cells became clearly visible, even with no stain penetrating into the cell.
4. Along the edge of the sample, where I cut the onion, I could see stain penetrating fully into each cell that was damaged by the cut and exposed to the stain.
5. In all the cells that were cut, and had stain within them, it appeared that the nucleus had exploded! Was this caused by osmotic action? I used tap water to flood the onion skin.
6. In the cells with what appeared to be exploded nuclei, there was a plethora of material which looked to have leaked out of the nucleus?

All in all a fun experience, and one I enjoyed greatly. It does leave me with some questions:

1. Is it normal to have the cover slip rock? The slides I used were beveled edge flat slides.
2. Is there a way to get the stain to penetrate into intact cells?
3. Once finished I assume I clean and reuse both the slide and cover slip?
4. I noticed that the stage movement controls on the Microscope are sluggish, is this normal?

I am amazed at how sharp things were, I even believe that at 100X I saw brownian motion on two very tiny bubbles that were next to each other. They were bouncing around in what appeared to be a random walk, while everything else was rock stable.

Any suggestions, hints, or comments would be appreciated...
Thanks and 73,
Dave
NK7Z
https://www.nk7z.net

MicroBob
Posts: 1489
Joined: Sun Dec 25, 2016 9:11 am

Re: First Stain, and mounting

#2 Post by MicroBob » Mon Jun 10, 2019 7:27 am

NK7Z wrote: 1. Is it normal to have the cover slip rock? The slides I used were beveled edge flat slides.
2. Is there a way to get the stain to penetrate into intact cells?
3. Once finished I assume I clean and reuse both the slide and cover slip?
4. I noticed that the stage movement controls on the Microscope are sluggish, is this normal?
Hi Dave,
you made a well documented experiment here and you have observed closely.
Here is a process description from Oliver KiM that differs in some important ways from you process: http://www.microbehunter.com/staining-o ... ll-nuclei/
1. Slide and cover slip are more or less planar. As soon as you put somthing in between them the cover slip can rock on the object separating these planar surfaces.
2. See Oliver's method
3. I clean and reuse in most cases. Sometimes right at the spot, sometimes I put glassware into a beaker "to be cleaned". When smeared with harmful sunstances like Aroclor I throw them into a beaker "glass rubbish". Other people throw away everything but the microscope itself. :shock: I don't like throwing away stuff - ask my wife :lol:
4. Viewed under magnification even a well serviced stage may appear somewhat sluggish. Do you have other microscopes in reach to compare? Maybe your microscope needs to be serviced. The slide should then move lightly, the damping should be in the knobs. For microscope servicing it is an advantage to have special damping grease available. If you can repair a bicycle you could service a microscope stage.

Bob

Hobbyst46
Posts: 2157
Joined: Mon Aug 21, 2017 9:02 pm

Re: First Stain, and mounting

#3 Post by Hobbyst46 » Mon Jun 10, 2019 8:00 am

NK7Z wrote:Hello,
Just did my first stain and mount. I carefully dissected an onion, removing a very thin peel of skin from the inside of the onion. Got it laid out on the slide, placed a small drop of water on it, then carefully put the cover slip on, by lowering it, starting at a 45 degree angle, and using a needle to lower it the rest of the way. Once finished, I added a drop of stain to one side of the cover slip, used a paper towel to suck the stain under and across the cover slip, such that the stain surrounded the onion skin.

I then did some cleanup on the slide, and let the stain soak while I cleaned up the dissecting station I built. Took about five minutes... Here is what I noticed:

1. Cover slip was being held above the slide surface by the onion skin, such that it could rock a bit. Is this normal?
2. The stain did not penetrate the onion skin very deeply. Just around the edges.
3. Many intact cells were clearly visible, and with some light diaphragm adjustment, the nucleus of the cells became clearly visible, even with no stain penetrating into the cell.
4. Along the edge of the sample, where I cut the onion, I could see stain penetrating fully into each cell that was damaged by the cut and exposed to the stain.
5. In all the cells that were cut, and had stain within them, it appeared that the nucleus had exploded! Was this caused by osmotic action? I used tap water to flood the onion skin.
6. In the cells with what appeared to be exploded nuclei, there was a plethora of material which looked to have leaked out of the nucleus?

All in all a fun experience, and one I enjoyed greatly. It does leave me with some questions:

1. Is it normal to have the cover slip rock? The slides I used were beveled edge flat slides.
2. Is there a way to get the stain to penetrate into intact cells?
3. Once finished I assume I clean and reuse both the slide and cover slip?
4. I noticed that the stage movement controls on the Microscope are sluggish, is this normal?

I am amazed at how sharp things were, I even believe that at 100X I saw brownian motion on two very tiny bubbles that were next to each other. They were bouncing around in what appeared to be a random walk, while everything else was rock stable.

Any suggestions, hints, or comments would be appreciated...
I would suspect that the onion skin you mounted was not the thinnest possible skin, but consisted of several layers, because otherwise, the stain would be rapidly and quite uniformly absorbed by most or all of the cells, not just along cell edge.

To peel off the really thin skin (bottom epidermis), I take a fleshy leaf from the bulb of a white or violet onion, and place it on the cutting board, convex side (shiny side) up. Then I carefully make a transverse incomplete cut in the middle of the slice, trying to leave both halves bridged to each other by the thin skin. I then hold the two halves (each with two fingers of one hand) and gently pull them apart, to seperate them from each other, without tearing. This motion usually peels the skin nicely.

Fixation of the skin prior to staining might help. I fixed them in IPA+lactic acid, 3:1, for a few minutes. It prevented the tendency of the skin to curl. I found hot water ineffective for this purpoose.

IMO, a stain concentration of between 0.02 and 0.05% or even 0.1% yields nice, not exaggerated staining within 5-10 minutes (or even less). 1% solutions produce too dark-colored cells.

I recycle slides. I recycle cover slips only if they are from a high-quality brand.

Tap water or stain solutions at the above concentrations are unlikely to have any significant effect on the osmotic pressure and should not explode onion cell nuclei due to osmotic effects.

Incidentally - which stain did you try ?

Note: Forum member mrsonchus is an expert on plant cell slide preparations.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

NK7Z
Posts: 18
Joined: Wed Jun 05, 2019 7:24 pm
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Re: First Stain, and mounting

#4 Post by NK7Z » Mon Jun 10, 2019 6:37 pm

MicroBob wrote:
NK7Z wrote: 1. Is it normal to have the cover slip rock? The slides I used were beveled edge flat slides.
2. Is there a way to get the stain to penetrate into intact cells?
3. Once finished I assume I clean and reuse both the slide and cover slip?
4. I noticed that the stage movement controls on the Microscope are sluggish, is this normal?
Hi Dave,
you made a well documented experiment here and you have observed closely.
Here is a process description from Oliver KiM that differs in some important ways from you process: http://www.microbehunter.com/staining-o ... ll-nuclei/
1. Slide and cover slip are more or less planar. As soon as you put somthing in between them the cover slip can rock on the object separating these planar surfaces.
2. See Oliver's method
3. I clean and reuse in most cases. Sometimes right at the spot, sometimes I put glassware into a beaker "to be cleaned". When smeared with harmful sunstances like Aroclor I throw them into a beaker "glass rubbish". Other people throw away everything but the microscope itself. :shock: I don't like throwing away stuff - ask my wife :lol:
4. Viewed under magnification even a well serviced stage may appear somewhat sluggish. Do you have other microscopes in reach to compare? Maybe your microscope needs to be serviced. The slide should then move lightly, the damping should be in the knobs. For microscope servicing it is an advantage to have special damping grease available. If you can repair a bicycle you could service a microscope stage.

Bob
Bob,
Thank you for the complement Bob, and thank you for the additional information as well... I suspect that the stage has older grease in it. The scope was made in the 70's, and I doubt it has ever been serviced. I may disassemble the stage and use White Lithium grease...
Thanks and 73,
Dave
NK7Z
https://www.nk7z.net

NK7Z
Posts: 18
Joined: Wed Jun 05, 2019 7:24 pm
Contact:

Re: First Stain, and mounting

#5 Post by NK7Z » Mon Jun 10, 2019 6:48 pm

Hobbyst46 wrote:
NK7Z wrote:Hello,
Just did my first stain and mount. I carefully dissected an onion, removing a very thin peel of skin from the inside of the onion. Got it laid out on the slide, placed a small drop of water on it, then carefully put the cover slip on, by lowering it, starting at a 45 degree angle, and using a needle to lower it the rest of the way. Once finished, I added a drop of stain to one side of the cover slip, used a paper towel to suck the stain under and across the cover slip, such that the stain surrounded the onion skin.

I then did some cleanup on the slide, and let the stain soak while I cleaned up the dissecting station I built. Took about five minutes... Here is what I noticed:

1. Cover slip was being held above the slide surface by the onion skin, such that it could rock a bit. Is this normal?
2. The stain did not penetrate the onion skin very deeply. Just around the edges.
3. Many intact cells were clearly visible, and with some light diaphragm adjustment, the nucleus of the cells became clearly visible, even with no stain penetrating into the cell.
4. Along the edge of the sample, where I cut the onion, I could see stain penetrating fully into each cell that was damaged by the cut and exposed to the stain.
5. In all the cells that were cut, and had stain within them, it appeared that the nucleus had exploded! Was this caused by osmotic action? I used tap water to flood the onion skin.
6. In the cells with what appeared to be exploded nuclei, there was a plethora of material which looked to have leaked out of the nucleus?

All in all a fun experience, and one I enjoyed greatly. It does leave me with some questions:

1. Is it normal to have the cover slip rock? The slides I used were beveled edge flat slides.
2. Is there a way to get the stain to penetrate into intact cells?
3. Once finished I assume I clean and reuse both the slide and cover slip?
4. I noticed that the stage movement controls on the Microscope are sluggish, is this normal?

I am amazed at how sharp things were, I even believe that at 100X I saw brownian motion on two very tiny bubbles that were next to each other. They were bouncing around in what appeared to be a random walk, while everything else was rock stable.

Any suggestions, hints, or comments would be appreciated...
I would suspect that the onion skin you mounted was not the thinnest possible skin, but consisted of several layers, because otherwise, the stain would be rapidly and quite uniformly absorbed by most or all of the cells, not just along cell edge.

To peel off the really thin skin (bottom epidermis), I take a fleshy leaf from the bulb of a white or violet onion, and place it on the cutting board, convex side (shiny side) up. Then I carefully make a transverse incomplete cut in the middle of the slice, trying to leave both halves bridged to each other by the thin skin. I then hold the two halves (each with two fingers of one hand) and gently pull them apart, to seperate them from each other, without tearing. This motion usually peels the skin nicely.

Fixation of the skin prior to staining might help. I fixed them in IPA+lactic acid, 3:1, for a few minutes. It prevented the tendency of the skin to curl. I found hot water ineffective for this purpoose.

IMO, a stain concentration of between 0.02 and 0.05% or even 0.1% yields nice, not exaggerated staining within 5-10 minutes (or even less). 1% solutions produce too dark-colored cells.

I recycle slides. I recycle cover slips only if they are from a high-quality brand.

Tap water or stain solutions at the above concentrations are unlikely to have any significant effect on the osmotic pressure and should not explode onion cell nuclei due to osmotic effects.

Incidentally - which stain did you try ?

Note: Forum member mrsonchus is an expert on plant cell slide preparations.
Thank you for the information on how to peal the onion, and on stain penetration! I only saw one layer of cells as I racked focus, so I assumed it was a sheet one cell wide. I will try that again later today. With regards to fixation, how long do you leave your sample in IPA? Also-- can I use IPA only for fixation? In my original process I did not perform any fixation! Where might one locate lactic acid?

The stain was "Eosin Y", and there is no dilution information which came with it. That coupled with the process I used, (water flush across cover slip), would have made even a guess at dilution difficult. The onion cells are sure large! :)
Thanks and 73,
Dave
NK7Z
https://www.nk7z.net

Hobbyst46
Posts: 2157
Joined: Mon Aug 21, 2017 9:02 pm

Re: First Stain, and mounting

#6 Post by Hobbyst46 » Mon Jun 10, 2019 8:34 pm

I have stained onion skin with several different dyes, but not Eosin. If time permits, I can try it, within a week or two.
I bought lactic acid at a local pharmacy, that specializes in "original" lotions and creams based on natural ingredients. A few ml, for a few $$.
Lactic acid is a slightly syruppy liquid, colorless, very slight pungeny odor. It is a weak acid, no special safety rules apart from the usual ones with any chemical - do not get it in the eyes, avoid skin contact, perhaps it is irritating somewhat.
Lactic acid is the stuff that accumulates in the muscles when they tire.
I left the onion skin in the IPA+LA for about 2 hours, probably 1 hour was sufficient.
Later, for the staining, I washed the skin in DW, since the stains were all in water solutions.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

NK7Z
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Re: First Stain, and mounting

#7 Post by NK7Z » Tue Jun 11, 2019 3:29 pm

Hobbyst46 wrote:I have stained onion skin with several different dyes, but not Eosin. If time permits, I can try it, within a week or two.
I bought lactic acid at a local pharmacy, that specializes in "original" lotions and creams based on natural ingredients. A few ml, for a few $$.
Lactic acid is a slightly syruppy liquid, colorless, very slight pungeny odor. It is a weak acid, no special safety rules apart from the usual ones with any chemical - do not get it in the eyes, avoid skin contact, perhaps it is irritating somewhat.
Lactic acid is the stuff that accumulates in the muscles when they tire.
I left the onion skin in the IPA+LA for about 2 hours, probably 1 hour was sufficient.
Later, for the staining, I washed the skin in DW, since the stains were all in water solutions.
Thank you for sharing your process... I will get some Lactic acid from a local supplier and give it a try! Also thanks for sharing the method as well! I am now looking for a decent starter camera, 5 MP or better. If you have any suggestions I am all ears...
Thanks and 73,
Dave
NK7Z
https://www.nk7z.net

Hobbyst46
Posts: 2157
Joined: Mon Aug 21, 2017 9:02 pm

Re: First Stain, and mounting

#8 Post by Hobbyst46 » Tue Jun 11, 2019 6:03 pm

NK7Z wrote:...I am now looking for a decent starter camera, 5 MP or better. If you have any suggestions I am all ears...
The 5MP USB camera cost me <$70 and is decent for documentation (see in your post "My New Microscope").
Forum member mrsonchus has had success with a Toupcam camera.
A very light-sensitive 14MP or 18MP (can't remember) USB camera has been recommended by Bradscopegems in this post:
viewtopic.php?f=9&t=7516&p=66342&hilit=astro#p66342.
I personally would hesitate to install a heavyweight DSLR camera (say, 0.5-1 Kg body) on one of the bino eyepiece tubes.
Many mirrorless cameras weigh only 0.3-0.5Kg (body).
Some doubt about the lack of aberration (chromatic, spherical etc) corrections when using any camera exists. If the camera is installed afocally, the weight of the lens also counts. Forum member MicroBob has posted his setup of a Nikon J camera onto a binocular tube.
If your choice is a DSLR or mirrorless camera, I would verify that it can be tethered to a PC such that shooting is controlled by software. Some cameras can be controlled through WiFi, IMO cable connection to PC is better.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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