Some Of My Homemade Permanent Slides

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desertrat
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Some Of My Homemade Permanent Slides

#1 Post by desertrat » Wed Dec 19, 2018 5:23 pm

I had got interested in microscopy around the 5th grade, and received a Lionel Porter microscope with accessories for Christmas. I looked at pond water, fly wings, and other usual things a beginner would look at. Getting into high school, I received a Tasco zoom microscope in a kit with slide making supplies for Christmas. The accessories included slides, cover slips, tiny plastic squeeze bottles of stains like eosin, methylene blue, safranin, fuchsin, and others. Sometime later I acquired a Perfect 809 slide preparation kit from the local hobby store. It had slides, coverslips, slide labels, and a small bottle of Canada Balsam mounting fluid.

I had checked out a book from the library titled Exploring With Your Microscope by Julian Corrington. It had instructions for making permanent slides. One of my early attempts was a piece of onion skin, stained with eosin from the microscope kit, and mounted in balsam from the slide making kit.

I don't remember the exact procedure I used, as I wasn't taking notes at the time. I didn't use a fixing fluid for the onion skin piece. Instead, it was sort of fixed by starting with 30% alchohol and going up through the alcohol series. But the time it got into pure alcohol, it was more or less fixed. I don't remember which alcohol I used, but it was probably isopropyl rubbing alcohol. I also don't remember at what point I stained the sample with eosin. It could have been before going into the alcohols, or further up the chain. I don't remember how I obtained alcohol water free enough to pass into xylene. The rubbing alcohol at hand was about 70%.

After the sample had soaked in xylene for a while, it was placed on a slide and mounted with the Canada balsam from the slide making kit. I think it took several attempts to get a usable permanent slide, but I don't remember how many. Below is the slide making kit.
Slide_kit.JPG
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Below is the kit opened up. The empty compartment in the lower left is for the bottle of balsam mounting fluid. Many years ago the blue plastic cap got brittle and broke. Underneath it was a hollow plastic plug pressed into the neck of the bottle to prevent evaporation, but it had become brittle. The bottle was removed to prevent balsam from leaking onto the slides and stored separately. After several moves of residence I have no idea of where it is. Some of the original blank slides are still there, the little square box with cover slips and labels, some missing slides, and permanent slides I made. The four slides on the right are antique prepared slides I bought on Ebay a couple of years ago. Perfect Parts Company is still in business, and still show this slide making kit on their website, but don't sell directly to consumers. They sell directly only to hobby dealers.
Slide_kit_open.JPG
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Below is the prepared onion skin slide. I think I was about halfway through high school when I made this.
Onionskin_eosin_slide.JPG
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Took this photo yesterday with the Polaroid digicam and a 10X compensating eyepiece for relay lens. The objective was an A/O Spencer apochromat 10X, .030 N.A. The slide has held up well for almost 50 years.
Onionskin_eosin.JPG
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Eosin isn't generally used for permanent slides of whole mounts, but is recommended by some websites for young people learning to make permanent slides. This one turned out not too badly. Carmine and hematoxylin generally give the best results for whole mounts, although carmine isn't generally used in botanical work.
Rick

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Hobbyst46
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Re: Some Of My Homemade Permanent Slides

#2 Post by Hobbyst46 » Wed Dec 19, 2018 5:37 pm

You did first-rate work as far as I see. I wish my present slides of onion skin would be so nice and clean when fresh...

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Re: Some Of My Homemade Permanent Slides

#3 Post by desertrat » Wed Dec 19, 2018 5:53 pm

Full disclosure: I showed one small good portion of the mount. Some other portions didn't preserve nearly as well. As I recall, the onion skin pieces seemed hydrophobic and water repellent, and wouldn't sink below the surface of water or weak alcohol mixtures. And it was impossible to keep air bubbles out of temporary mounts of fresh material. The onion skin pieces began to sink when going into the stronger alcohol mixtures, and that got rid of the air bubbles.

I haven't tried it, but a wetting agent in the water might help, like a very small amount of dish washing detergent, or the wetting agent added to dish washing machines. A photographic wetting agent like Photo-flo 200 might work also.
Rick

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Re: Some Of My Homemade Permanent Slides

#4 Post by Hobbyst46 » Wed Dec 19, 2018 6:04 pm

I would think that what counts in our case (hobby microscopists who do non-profit work) is not the percentage of well mounted samples, but rather the best slide that one can make. So we are not bound by statistics.

Onion skins fold and roll up and float today, like they did 20 years ago. So, before staining, I fixed them with alcohol and lactic acid, which eliminated the problem more or less. I also tried warming them up to 60C (as far as I remember - could be wrong) as suggested by the late Walter Dioni, but that was futile.

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Re: Some Of My Homemade Permanent Slides

#5 Post by apochronaut » Thu Dec 20, 2018 1:11 am

Pretty nice slide imo. ..especially for 50 years ago. I have a Perfect dissecting kit, in a very similar wooden box.

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Re: Some Of My Homemade Permanent Slides

#6 Post by desertrat » Thu Dec 20, 2018 2:51 am

Thanks, apo! I got one of those dissecting sets in grade school, still have it. The cool little glass applicator rod was really neat. Half blue cobalt glass, half clear glass, tapered toward each end with the ends neatly rounded. Until recently I was using it for applying small drops of castor oil or cedar oil to slides for viewing under oil immersion.

Then I dropped it and broke one end off. I was kind of bummed about that. Had it all these years, through multiple moves, then broke it in a moment's carelessness. I guess one shouldn't get overly attached to these things, but I went on Ebay and soon found another Perfect kit of the same type and bought it. The new glass rod was a bit smaller than the old one, but otherwise the same. In a matter of months, I dropped that one and broke it too.

Now I'm back to using the original damaged one, the clear glass side is intact, and keep it in a small glass jar stuffed with paper towels. I now have plenty of the slightly crudely made scalpels, forceps, needles, magnifier, etc. Don't need to buy another set.

Here's another of my early attempts to make a permanent slide. Scraped some cheek cells off the inside of my mouth with a toothpick and smeared them on a cover slip. Then the cover slip was passed through the flame of an alcohol lamp to fix the smear. After it cooled, put a couple drops of methylene blue from the Tasco microscope kit on the smear. After a couple of minutes, rinsed that off and let the cover slip dry. Then mounted in balsam.
Cheek_cells_slide.JPG
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Some of the cheek cells got kind of cooked from the flaming treatment, but found a group of them on the slide that don't look too bad. The blue ovals are the cell nuclei, the large very faint blue blobs are the cytoplasm, and the small short rods are bacilli. Used the little digicam with 10X compensating eyepiece for relay lens, and 43X 0.95 N.A. Apo objective.
Cheek_cells.JPG
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Rick

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Re: Some Of My Homemade Permanent Slides

#7 Post by MicroBob » Thu Dec 20, 2018 9:26 am

Hi Rick,

thank you for showing your old slides, they are very nice!
I like permanent slides very much but never had the means to make some when I was a boy.
When it comes to perfectly looking slides I always think about Robert Koch. A member of our group once had a look at the original slides of him and made some micro photographs of them too. Robert Kochs slides mostly looked as if he had made them in the dark behind his back after a bottle of Eierlikör - and he became famous for what he found out with them. :lol: This made me very relaxed about the looks of my slides!

Bob

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Re: Some Of My Homemade Permanent Slides

#8 Post by desertrat » Thu Dec 20, 2018 3:55 pm

Thanks, Bob! I've been making permanent slides off and on since then, with pauses of several years between slide making sessions, sometimes many years. My most recent batch was made at the end of last March. Still haven't put labels on them yet, have been very lazy about that. I need to get them labelled before I forget what I did...
Rick

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Re: Some Of My Homemade Permanent Slides

#9 Post by MicroBob » Thu Dec 20, 2018 5:22 pm

Hi Rick,
very nice for labeling slides: The Brother PT2430PC label printer. It can print 24mm labels with a lot of fine but readable print on it.
The more slides of the same content you have, the more useful it becomes.

Bob

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Re: Some Of My Homemade Permanent Slides

#10 Post by desertrat » Sun Dec 23, 2018 3:23 am

Another stained onion skin slide. A year of so after I made the first slide, my parents kindly ordered some chemicals for fixing, staining, and mounting permanent slides from a company that advertised in Popular Science, Popular Mechanics, and other similar magazines, and sold these products to private individuals. The shipment contained some chemicals for fixatives, bottles of prepared stains, alcohol, some acids, xylene, etc.

Among the stains were little one ounce (about 25ml) bottles of Delafield's hematoxylin, Ehrlich's hematoxylin, Borax Carmine, Wright's blood stain, and some others. I still have most of the bottles from that first shipment, almost 50 years ago, although most of the stains have dried up. The cardboard gaskets in the stamped steel screw caps weren't completely vapor tight.

A small amount of CRAF fixative was mixed from instructions in Corrington's microscopy book, based on the formula of Navaschin's fixative, containing formalin, acetic acid, and a small amount of chromic acid. The piece of onion skin was fixed in that for a short time, maybe about a hour, then transferred to several changes of water. It was then placed in Delafield's hematoxylin, I don't remember if it was full strength or diluted with water, probably for a short time, less than an hour.

After several changes of water to rinse out the excess stain, the piece of onion skin was dehydrated through a series of alcohols, then to xylene, then mounted in balsam from the slide making kit.
On_skn_hmtxln_slide.JPG
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I only put labels on the first few slides I made. After that I didn't bother anymore. A bad habit, I know. I didn't start labeling slides again until just a few years ago. :oops: At the time, I figured I could remember anything important about how the slide was made. And I probably did remember, for several years, but not 50 years. Below a view of the onion skin, 10X apo. N.A. 0.30, digicam with 10X compensating eyepiece relay lens.
On_skn_hmtxln2.JPG
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Fixation and stain is more even, and more details can be seen in the cell nuclei. The nucleoli can be seen in both slides, but the granular chromatin is visible only in the second slide, probably due to better fixation and better stain. The first onion skin specimen wasn't really fixed, just dehydrated in the alcohol series. Hematoxylin is a good nuclear stain, whereas eosin is generally only used as a counterstain for the cytoplasm. An inset from a section of the original image, unreduced in size.
On_skn_hmtxln2_inset.JPG
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The tiny granules of chromatin in the nuclei can be seen more clearly through the eyepiece. The image is a bit fuzzy because it's equivalent in size to looking at the objective with about a 30x eyepiece.

The little Polaroid digicam was behaving a bit better today. I worked out some focus issues. Turns out there is a small focus error between the vertical tube and the two inclined tubes. I don't want to trim the vertical tube, and I could put shims on the ends of the two inclined tubes, but for now I'm just making focus adjustments before making the exposure.

Previous images had illumination fall-off at the outside of the image circle, even though illumination appeared even through the eyepieces. Turns out for this application, the illumination circle on the slide has to be much bigger than what is required to fill the field in the eyepieces. To do this I replaced the achromatic condenser, which has kind of a small illumination circle, with the Abbe condenser with the top element removed. The condenser focus was adjusted until the stopped down field iris was focused at the specimen plane, then the field iris was opened all the way up. This wouldn't be ideal for getting maximum performance from the objective, but makes for more even illumination when using the camera.

After this slide, I attempted to do a double stain with hematoxylin and eosin, but my efforts to differentiate the stain weren't successful, and the double stained slides looked more or less like this one that was stained only with hematoxylin. A trace of eosin could be seen on the only other slide I kept.
Rick

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Re: Some Of My Homemade Permanent Slides

#11 Post by desertrat » Sat Jan 05, 2019 12:00 am

After these early slide making attempts in my late teens, many years went by without using the microscope.

A little over ten years later, in the mid 1980s, I started looking at samples of pond water from a local park. Attempts were made to make some permanent slides of spirogyra, stained in hematoxylin and mounted in balsam, but the stain wasn't very good and the strands of algae collapsed in the xylene. No permanent slides were made. After a few months, microscopy went on the back burner again.

About 10 years later, in the late 1990s, started collecting samples of pond water and growing cultures again. Obtained samples of aquatic annelid worms, Chaetogaster, Nais, and Pristina. There were also planarian flatworms, microcrustacea, and hydra.

Using books acquired on making permanent slides, started processing samples to make permanent slides. During that time the efforts were mostly failures, but there were a few somewhat successful stained mounts in balsam. The few successes gave enough satisfaction to keep working at it. There was no mentor to show the details of how it should be done, so a lot of different things were tried. I discovered dozens of ways to lose specimens going through the stages of fluids, or have specimens fall apart or shrivel up from using the wrong fluids or process the specimens through the wrong order of fluids. I learned many, many different ways of how *not* to process specimens.

My first successes were with tiny annelid worms, later flatworms and hydras, and a few successes with the microcrustacea.

I wanted to present the successful slides more or less in chronological order, but after getting images of the first successful hydra slide, I realized my memory had been playing tricks on me, and this slide came several years after the first successful slides of the annelids.

The slide below was made probably about 15 years ago. At that time I had a pretty good collection of chemicals for fixatives, stains, dehydration, and mounting. But I was interested in seeing if slides could be made from products obtained from commercial sources, not lab supplies companies.

The fixative was a 2% solution of copper sulfate in water. There was mention in an early edition of The Microtomist's Vade-Mecum about copper sulfate used as a fixative for Siphonophora, relatives of jellyfish. Hydras are distant relatives of jellyfish, so maybe the copper sulfate would work. Here and where I used to live, 2 lb bottles of copper sulfate are available at the nearest hardware store, used to kill roots clogging drain lines leading to the sewer.

A hydra was dislodged from the wall of the container with a needle and wide mouth pipette with minimal damage to the holdfast at its base. It was then dropped into the fixing solution quickly before attaching itself to the inside of the pipette. The second part of this process was only successful after several tries. Hydras can attach themselves to the inside of the pipette very quickly.

The hydra began to contract, but "froze" after only a second or so, before it could contract completely. It was fixed, at least enough so it couldn't contract any more.

After a few minutes it was transferred to the stain. Some stain recipes from the 1880s mentioned making hematoxylin stains from logwood shavings, and some of these had been bought online from a vendor of organic fabric dyes. The usual mordant was alum, but some of the early recipes used other mordants containing copper, calcium, chromium, etc. The local hard tap water contained plenty of calcium compounds, so a stain was made up from logwood shavings soaked in the hard tap water. At the time, I was doing lots of hare-brained experiments like this. Trying all kinds of odd things just to see if any would work.

Earlier attempts to stain with this didn't work very well, the stain was very faint. This time, instead of using a vial, some stain was poured into a shallow dish, barely deep enough to cover the hydra, which was placed in the stain. I'm guessing the large surface contact with air made a difference, because after a minute or so the hydra seemed stained pretty well.

It was transferred slowly through the ladder of increasing concentrations of alcohol, ending up in concentrated alcohol.

Don't remember exactly what clearing agent was used, but it was probably xylene. From there is was carefully sucked up into the wide mouth pipette and gently squeezed out onto the slide. The excess xylene was carefully blotted up, a large drop of canada balsam added, and a coverslip carefully lowered on top.

For the first time, the slide mount contained a whole hydra, not broken up pieces of hydra. They tend to get brittle when passed through fluids containing no water. Only one tentacle was partially broken off at the base. It was my first successful hydra slide.

The image below was taken with the little digicam using the 10x compens eyepiece, objective was 3.5X N.A. 0.08. The abbe condenser was used with the top lens removed. Leaving the top lens on and using the swingout lens at the bottom of the condenser doesn't give even illumination across the field.
Hydra1_a.JPG
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The next image is a closeup of a tentacle. The arrows point to a couple of discharged nematocysts. The everted nematocyst tube can be seen near the lower arrow, sticking out past the tops of the other nematocysts. The barbs are barely visible part way up the tube, looking like a tiny thin crossarm on a tiny telephone pole. The long thread coming off the end can be seen drifting away and going out of focus. A length of thread from another nematocyst can also be seen. The hydra managed to fire off quite a few nematocysts after it went into the fixer. Objective was a 43X achromat.
Hydra1_b1.jpg
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The last image is a closeup of the side of the hydra's body, just below the tentacles. The margins of the squamous cells on the outer surface of the hydra's ectoderm can be seen. A few cell nuclei can also be seen, faint and barely darker than the cytoplasm of the cells. A properly applied hematoxylin stain, properly mordanted, can show these details of where cell membranes separate cells from adjoining cells. This feature being visible was a lucky accident. I had no idea it would be visible. Objective was 20X N.A. 0.5 achromat. I have a 20X N.A. 0.6 apochromat, better for photography, but it works very close to the slide. Some of my wholemounts are too thick to use it. The N.A. 0.5 achromat used for this image is a brass antique, more than a century old. It gives such a good image I have made no attempt to acquire a more modern 20X achromat.
Hydra_epithelium_sm.JPG
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Edit to add, the above image was taken with the little 2 MP eyepiece camera held on top of the new 0.3X reducing lens that just came in. It seems to work pretty well. Will need to make an adapter to hold them together. The reducing lens has an upper C-mount thread for a C-mount microscope camera. When they are mounted together, the little digicam won't need to be used much anymore, and the eyepiece camera can be used with other scopes that don't have trinocular heads.
Rick

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Re: Some Of My Homemade Permanent Slides

#12 Post by MicroBob » Sat Jan 05, 2019 8:02 pm

Hi Rick,
thank you for posting your preparation method for the hydra! I like experimental microcopy methods a lot and I also think that they are very worthwhile: When you look what problems microscopists, especially beginners have, you frequently find that one of the main problems is that they know the common recipes, but can't use them at home. This can be because of a lack of access to chemicals, because of rare chemicals, expensive chemicals, harmful chemicals, waste problems or lack of lab equipment. And here in the forum alternative preparaion methods seem to be of interest for many members.

In my plancton aquarium there are lots of green hydras - If I find the time I certainly will give it a try.

Bob

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Re: Some Of My Homemade Permanent Slides

#13 Post by einman » Sun Jan 06, 2019 1:54 am

All in All quite nice!

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Re: Some Of My Homemade Permanent Slides

#14 Post by desertrat » Sun Jan 06, 2019 2:08 am

Thanks, Bob!

Over the years I have done research on internet based specialty supply companies, that sell to the public, that have many of the chemicals used in the older recipes for permanent slide making. I don't know how available these products would be outside the US.

Some chemicals can also be obtained at supermarkets, hardware stores, auto parts stores, etc. Once again, I don't know how many of these are available outside the US.

I you want, in the next few days I can post a list of places where most of the needed chemicals and dyestuffs can be found that aren't lab supply companies.

Here's a brief sample:

It seems in the mid 19th century, microscopists discovered the resins used for mounting permanent slides in the workshops were lacquers and varnishes were being used to finish furniture and musical instruments. Canada balsam, dammar gum, rosin (colophony), all those resins can be used for mounting specimens.

Turpentine used by artists and painters can be used as a clearing medium the same way xylene is used. I've used it, and it works, but I prefer xylene for other reasons. One of main ones is I can buy gallon cans of xylene and toluene at the local hardware store, and it doesn't change over time the way turpentine does.

Arthur Bolles Lee wrote that special thickened turpentine was popular as a clearing medium in Germany in the late 19th century. I believe thickened turpentine was used by musical instrument makers before it was discovered by microscopists.

There is a company in Germany that sells many of these products, including logwood and cochineal to make stains, Kremer Pigmente. I don't know if they sell to individuals, however.

Anyway, there is a lot more to come on this subject if anyone is interested in making permanent slides with these products.

Edit to add, also thanks einman!
Rick

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Re: Some Of My Homemade Permanent Slides

#15 Post by MichaelG. » Sun Jan 06, 2019 8:58 am

desertrat wrote:Over the years I have done research on internet based specialty supply companies, that sell to the public, that have many of the chemicals used in the older recipes for permanent slide making. [ ... ] I you want, in the next few days I can post a list of places where most of the needed chemicals and dyestuffs can be found that aren't lab supply companies.
That would be very useful, Rick

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Re: Some Of My Homemade Permanent Slides

#16 Post by MicroBob » Sun Jan 06, 2019 11:40 am

Hi Rick,
I'm sure it would be very useful if you could post this list here. I would not include stuff the supplyer isn't supposed to sell to the local public into a public available list though. There have been examples where limitations have not been cared for and big problems resulted from it, not only for the shop.

My impression is that microscopists in earlier times often had unlimited access to chemicals and also didn't care much for safety and environment. They were doing their preparation and left and right peopl, animals and plants started to dwindle. :lol:

There are several recipes that use narcotics or very dangerous substances. Also costs can sum up to high amounts when you have to buy much more substance than you need for microscopical purposes. I'm always especially satisfied when I can present a method that works with commonly available substances and is so cheap that nearly everybody could use it. There are many scientifical interested people around the world with very limited access to whatever stuff and very limited financial means compared to Europe and the USA. I like to include everybody into the hobby.

Bob

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Re: Some Of My Homemade Permanent Slides

#17 Post by Hobbyst46 » Mon Jan 14, 2019 8:38 pm

I re-inspected some onion upper epidermis slides that I had made 9 months ago. They had been fixed with IPA-lactic acid, 3:1, stained with Safranin, Or Methylene Blue, or Toluidine Blue, mounted in 80% fructose in water and immediately sealed with nail polish. Initially they looked well. Today, they have deteriorated. Although the seal has been leak proof, no evaporation of water, the stain was washed out, diffused away from the skin specimen to the edge of the coverslip. Safranin stained look better, but still not the same as when new. Perhaps I was supposed to wait, between the mounting and sealing, to let the fructose mountant to dry out somewhat and solidify; anyway, I am guessing that PVA might be better than fructose, as suggested a few weeks ago by mrsonchus from his experience.

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Re: Some Of My Homemade Permanent Slides

#18 Post by desertrat » Mon Jan 14, 2019 8:51 pm

Hobbyst46 wrote:I re-inspected some onion upper epidermis slides that I had made 9 months ago. They had been fixed with IPA-lactic acid, 3:1, stained with Safranin, Or Methylene Blue, or Toluidine Blue, mounted in 80% fructose in water and immediately sealed with nail polish. Initially they looked well. Today, they have deteriorated. Although the seal has been leak proof, no evaporation of water, the stain was washed out, diffused away from the skin specimen to the edge of the coverslip. Safranin stained look better, but still not the same as when new. Perhaps I was supposed to wait, between the mounting and sealing, to let the fructose mountant to dry out somewhat and solidify; anyway, I am guessing that PVA might be better than fructose, as suggested a few weeks ago by mrsonchus from his experience.
The stains you used are popular for use in regular microtechnique, in which all the water in the specimens is gradually removed and replaced by other fluids, which are eventually replaced by a resinous mounting medium containing no water.

I'm guessing some of these stains will not keep over long periods in an aqueous mount. I think there are some stains that can be used in an aqueous mount, but don't know which ones they are off hand.
Rick

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Re: Some Of My Homemade Permanent Slides

#19 Post by Hobbyst46 » Mon Jan 14, 2019 9:03 pm

desertrat wrote:
Hobbyst46 wrote:I re-inspected some onion upper epidermis slides that I had made 9 months ago. They had been fixed with IPA-lactic acid, 3:1, stained with Safranin, Or Methylene Blue, or Toluidine Blue, mounted in 80% fructose in water and immediately sealed with nail polish. Initially they looked well. Today, they have deteriorated. Although the seal has been leak proof, no evaporation of water, the stain was washed out, diffused away from the skin specimen to the edge of the coverslip. Safranin stained look better, but still not the same as when new. Perhaps I was supposed to wait, between the mounting and sealing, to let the fructose mountant to dry out somewhat and solidify; anyway, I am guessing that PVA might be better than fructose, as suggested a few weeks ago by mrsonchus from his experience.
The stains you used are popular for use in regular microtechnique, in which all the water in the specimens is gradually removed and replaced by other fluids, which are eventually replaced by a resinous mounting medium containing no water.

I'm guessing some of these stains will not keep over long periods in an aqueous mount.
"Solid" PVA glue contains water, so these stains will be washed away in PVA too... do you think that the resin should not contain water at all ? for example, even the popular glycerine jelly contains water.

desertrat
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Re: Some Of My Homemade Permanent Slides

#20 Post by desertrat » Mon Jan 14, 2019 9:48 pm

Hobbyst46 wrote:"Solid" PVA glue contains water, so these stains will be washed away in PVA too... do you think that the resin should not contain water at all ? for example, even the popular glycerine jelly contains water.
Arthur Bolles Lee wrote that Carmine stains would keep in glycerine or glycerine jelly mounts.

Carminic acid, Carmine, and Cochineal can be used to make related stains. Carminic acid is the pure dye, extremely expensive and not sold by hobbyist suppliers.

Carmine contains about 50% carminic acid and some metal salts of aluminum and calcium which act as mordants. Carmine dye can be found on Ebay. The microscope dye carmine is rather expensive. There is an Ebay seller called "Cosmetic Maker" selling what appears to be real carmine and is used in cosmetics, and is far less expensive. I bought some several years ago and made up a batch of Grenacher's alcoholic borax carmine with it. I haven't used it yet on specimens, but after mixing it up it looks like "the real deal".

Cochineal is the raw material that carmine and carminic acid are made from. It is dried insect bodies, and is available from internet dealers in "organic" fabric dyestuffs. One brand I've seen on Ebay is Jacquard cochineal. Cochineal can be used for some carmine stains, those that contain mordants like alum added to the stain. Alum cochineal is a water based stain and one I've used a lot in the past. Cochineal can also be used to make a variation of Mayer's Paracarmine, which is dissolved in 70% ethanol. I've used that with good results, also. I don't know if IPA will work in this formula.

I still think there are a few modern, synthetic stains that will work with aqueous mounting media. Apparently, aqueous mounting media are used with some fluorescence stains. I also saw mention that Crystal Violet might work with these media.
Rick

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Re: Some Of My Homemade Permanent Slides

#21 Post by Hobbyst46 » Mon Jan 14, 2019 10:05 pm

Thanks!
I will read about the carmine dyes and Crystal Violet. Almost sure I have CV somewhere. In addition, I might still try and make the most of classical organelle-specific dyes like toluidine blue, safranine etc, that so beautifully and selectively stain plant parts, with PVA as mountant.

desertrat
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Re: Some Of My Homemade Permanent Slides

#22 Post by desertrat » Mon Jan 14, 2019 10:22 pm

Hobbyst46 wrote:Thanks!
I will read about the carmine dyes and Crystal Violet. Almost sure I have CV somewhere. In addition, I might still try and make the most of classical organelle-specific dyes like toluidine blue, safranine etc, that so beautifully and selectively stain plant parts, with PVA as mountant.
Have you thought about using clear nail polish as a mountant? Did you mention nail polish in a previous post? My memory isn't very good, so it might be another member.

The solvent for nail polish is acetone, which happens to miscible with water in all proportions. Acetone has been used as a dehydration liquid in the past, using a graded series of water and acetone, similar to the alcohol series. Once the specimen is in pure acetone, or the nail polish remover if that's all that's available, it can then be mounted in the nail polish.

If your specimens come out of the stain in a solution of IPA, you can make a graded series of IPA and acetone (I think, not completely sure about this one). Once the specimen has been mounted in nail polish, all the water is gone, and the stain shouldn't fade. (Maybe).
Rick

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Re: Some Of My Homemade Permanent Slides

#23 Post by Hobbyst46 » Mon Jan 14, 2019 11:13 pm

Yes, the solvent of nail polish is either acetone or methyl ethyl ketone; both of them, IPA and water are all miscible among themselves, and I have access to all of them. However, whether the classical dyes, adsorbed on the plant, are resistant to these solvents (acetone, say) or are desorbed from the plant, I do not know. In addition, I used the stains in aqueous solution, not in organic solvent.

So if all else fails, I might try the alternative path that leads to nail polish as mountant. I used NP in the past, for dry plant parts, but did not like it, because of the air bubbles.

desertrat
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Re: Some Of My Homemade Permanent Slides

#24 Post by desertrat » Thu Feb 14, 2019 7:40 pm

Sometime in late 2004, I succeeded in getting another hydra specimen more or less intact in a permanent mount. This time the hydra was fixed in a hot solution of 2% copper sulfate with about 1/2% to 1% acetic acid derived from distilled white household vinegar which is 5% acetic acid. The hot solution fixed it more less instantly, so it contracted very little compared to the previous hydra slide.

After being fixed for a couple of hours, the hydra was transferred to a 5% solution of agricultural grade aluminum sulfate, which is a good short preservative and also the mordant of the following hematoxylin stain. After a couple of hours in this solution, the hydra was transferred to the hematoxylin stain which was based on an old recipe known as Cook's hematoxylin. It used mainly aluminum sulfate and a small amount of copper sulfate as a secondary mordant. The hematoxylin was extracted from some logwood shavings. This stain did a much better job than the experiment with hematoxylin mordanted with hard water in the previous hydra slide.

The hydra was then transferred through a series of increasing concentrations of alcohol solutions. From the highest concentration of alcohol, nearly 100%, the hydra was transferred to a couple of changes of turpentine from the local hardware store as a clearing agent. The first transfer was made into a layer of the alcohol pipetted carefully on top of a layer of turpentine to make a two layer solution. This allowed a gradual transfer of alcohol into turpentine in the hydra without causing any strong diffusion currents that could damage the hydra.

The hydra was then placed onto a slide with a couple of drops of turpentine, which the excess was carefully blotted away with small strips of newspaper margins. Then a couple of small drops of Canada balsam dissolved in xylene were dropped on top of the hydra, and a coverslip carefully lowered on top to complete the mount.

The mount was carefully placed on a level stage and observed with low power to see if it was any good. It was then placed in an out-of-the-way level place to cure somewhat before being looked at with higher power, probably a week or so.

This image was taken with the 2MP eyepiece camera with 0.3X reducing lens, using an antique brass Spencer 4X objective. Apologies for the banding. I have equipment on hand to feed the 4 Series lamp in the base with smooth, ripple free 6 volts DC. That will eliminate the banding completely. But I haven't made the connectors to hook it up yet. The portions of the image that look very dark, almost black, are some sort of artifact produced by the simple webcam image capturing software on the computer that the camera is connected to. These do not look nearly as dark seen through the eyepieces. This seems to happen mostly when there is lots of clear background in the image. This may have been aggravated by removing the condenser when using such low power objectives, otherwise the outer portions of the field look dark in the images, even though they look evenly illuminated in the eyepieces.
Hydra_2_4X.jpg
Hydra_2_4X.jpg (44.69 KiB) Viewed 13984 times
The following image shows a tentacle and the upper part of the main body with an antique brass Spencer 10X objective.
Hydra_2_10X_b.jpg
Hydra_2_10X_b.jpg (70.38 KiB) Viewed 13984 times
The following image shows a tentacle with an antique Spencer 20X objective. The portion with the upper surface in focus is looking at a nematocyst from directly above. The tip of the arrow is touching the outside of the cell that surrounds the nematocyst. When viewed from above, the nematocyst should be round, and did look that way when the slide was prepared. Unfortunately, within a few weeks after preparation of the slide, the nematocysts began to shrivel up. Some structures don't preserve their original shape well in balsam. The rest of the hydra's structures have not deteriorated noticeably in the last 15 years. The nematocyst is surrounded by smaller cells that form sort of a group of cells that many of the nematocysts form the center of.
Hydra_2_20X.jpg
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The following image shows the same tentacle with the focus adjusted into the middle of the tentacle. The tip of the left arrow is touching the lower margin of one of the cells making up the surface of the tentacle, viewed from the side. The tip of the middle arrow is touching a cell surrounding a nematocyst, seen from the side. The tip of the right arrow is touching the margin between two cells making up the outer surface of a nearby tentacle. These lines can be seen on some of the other images.
Hydra_2_20X_b.jpg
Hydra_2_20X_b.jpg (42.21 KiB) Viewed 13984 times
Rick

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Re: Some Of My Homemade Permanent Slides

#25 Post by desertrat » Thu Feb 14, 2019 7:55 pm

Please also see the previous post, which has the first four images of this set.

The following image, also taken with the antique brass 20X objective, shows the nuclei of two cells with the focus adjusted a little below the surface of the tentacle at these cell's location
Hydra_2_20X_c.jpg
Hydra_2_20X_c.jpg (44.31 KiB) Viewed 13983 times
The following image shows the upper surface of the outer layer of cells, the ectoderm, near the base of the hydra. Also brass 20X objective. The arrow points to the inside of a nematocyst that stained strongly purple. Copper sulfate when used as a mordant of hematoxylin gives this purple color versus the blue-violet color when a salt of aluminum is used. I'm guessing that a few of the nematocysts that stained deep purple like this, somehow absorbed a lot of copper sulfate, from either the fixer or the Cook's hematoxylin. Why only a few stained like this I have no idea.
Hydra_2_20X_d.jpg
Hydra_2_20X_d.jpg (56.88 KiB) Viewed 13983 times
The following image is focused a little deeper than the previous image, showing the lower layer of cells making up the endoderm of the hydra. The arrow is pointing at a small piece of the upper ectoderm that peeled away from the lower endoderm. I think the ectoderm is a good deal thinner than the endoderm. Looking at the left and right margins of the image, it's possible to see where the outer edges make up the ectoderm, although not very clearly. Many cell nuclei can be seen near the external margins of the endoderm cells.
Hydra_2_20X_e.jpg
Hydra_2_20X_e.jpg (53.92 KiB) Viewed 13983 times
Rick

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Re: Some Of My Homemade Permanent Slides

#26 Post by Hobbyst46 » Thu Feb 14, 2019 9:10 pm

I like this series of images, a step by step introduction to the structure of the animal, with emphasis is on the findings rather than on the prestige or high class of the equipment.

desertrat
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Re: Some Of My Homemade Permanent Slides

#27 Post by desertrat » Thu Feb 14, 2019 11:32 pm

Hobbyst46 wrote:I like this series of images, a step by step introduction to the structure of the animal, with emphasis is on the findings rather than on the prestige or high class of the equipment.
Thanks.

No point in me trying to pretend my equipment has prestige. Everyone here knows I'm using old stuff bought cheaply. It's too bad I can't get decently sharp images out of my eyepiece camera and reduction lens. My antique objectives produce much better images in the eyepieces than what these images show.
Rick

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Re: Some Of My Homemade Permanent Slides

#28 Post by MicroBob » Sat Feb 16, 2019 7:45 pm

Hi Rick,
these images are quite nice in fact.
The only things I don't like are the banding and a slight lack of apparent sharpness.

Do you have an idea where the banding comes from? The light source or the camera? Does changing the brightness of the lamp influence the banding?
Do you use unsharp masking in your image editing?

Bob

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Re: Some Of My Homemade Permanent Slides

#29 Post by desertrat » Sat Feb 16, 2019 8:29 pm

Hi Bob, thanks for the compliment.

The banding is coming from the 60 Hz fluctuations in brightness by the built in illuminator lamp filament being powered by AC. I could eliminate it completely by feeding the lamp with smooth DC. I succeeded doing that in an experiment I posted awhile back. But the image capture software I just downloaded from the website identified by Hobbyist46 has reduced the banding a lot, enough that I probably won't bother setting up a DC power supply for the lamp filament.

The microscope eyepiece camera doesn't appear to be able to produce really sharp images. I am sharpening them somewhat in GIMP using the unsharp mask tool, but if I go too far then strange artifacts appear like a grainy background, small dark details becoming completely black, and so on.

It looks like if I want to get decently sharp images, I will have to buy a better image capture device. Eventually I probably will, but don't have any near term plans at the moment.
Rick

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Re: Some Of My Homemade Permanent Slides

#30 Post by Hobbyst46 » Sat Feb 16, 2019 9:21 pm

Hi desertrat,
I added a note about Color space setting and image resolution in MICAM, in the "Inexpensive USB 2.0 camera test" post.

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