A new (?) home-made mounting medium for diatoms

[MichaelG.]

here: http://www.mikrohamburg.de/Tips/DER%20B ... SINDEX.pdf you can find a method for determining refractive indices. It's written in german ...

Bob

Many thanks for the link, Bob

MichaelG.

[MicroBob]

You're welcome, MichaelG!

Diatoms don't have all the same refractive index and their structures are different. So the necessary refractive index of the mounting medium will vary too. For some diatoms a r.i. of >1,65 is really needed to get a good contrast, e.g, Amphipleura Pellucida. But for many diatoms something like Caedax is sufficient. So not having Naphrax or Pleurax should't stop one from trying to get a good diatom image. BTW air has a very different r.i. from diatoms!

Here is a very interesting study on the long term stability of mounting media. Apart from the value of the experience of these researchers it is an interesting guide to the different mounting media: http://www.mapress.com/j/zt/article/dow ... .1.1/12414

[Hobbyst46]

MicroBob and Michael G - Thanks for the info and links.

Actually, some time ago I read the Rosenfeldt methods of RI measurement with self-made "refractometers". I was enthusiastic and tried to follow, by making the first two devices (relatively simple) that Rosenfeldt suggested. I failed. With one device - a razor blade attached to glass - I could not focus in the way that he suggested. Perhaps I misunderstood the Google translation from German. So I gave it up.

I hope to be read the article about mounting media that you linked to. Long term stability is a challenge. Right now, the PS-CBO should be considered a semi-permanent mountant at best.

Anyway, to demonstrate this mountant, I would like to post some brightfield images as well. These should be more informative in this context than the phase contrast images.

[Hobbyst46]

Thanks MicroBob again for the link http://www.mapress.com/j/zt/article/dow ... .1.1/12414

Now I really must make time to read it - I expected an article, this is a treatise!! and appears to be very comprehensive.

[einman]

Not sure if this was mentioned but optical adhesives have a refractive index of 1.56, although they are not cheap. They cost about the same as other highly quality mounting fluids. There are several High Refractive index silicones which are used to impart shine to hair etc. I have been looking at them. The coverslip needs to be sealed via fingernail polish etc. to prevent creeping of the silicone.

[Hobbyst46]

einman
Optical glass adhesives were indeed mentioned. Norland products like Norland 61 and others. They are based on nano zirconia, as far as I know, and that imparts the high RI. Hair sprays sound interesting! However, chemicals that boast a high RI are mostly hazardous, like bromo-naphthalene, they can be polyaromatic and toxic - I doubt that such substances will be allowed to touch the skin of hair.

[Micro-Bob]

Hi Hobbyst46,

I have not tried the measuring methods myself but I'm shure Mr. Rosenfeld has. If we could narrow the problem down I could ask him when I see him next time.

The refractive index of CBO can be found here:
https://www.sigmaaldrich.com/Graphics/C ... RICH__.pdf
It probably varies a little as it is a natural product.

Polystyrene is about the same:
https://refractiveindex.info/?shelf=3d& ... cs&page=ps

At nearly 1.6 the refractive index is high enough for most diatoms. For diatoms with heavy structures it might even give an especially nice image.
A chemist might have an idea whether this mountant is prone to crystallisation or other aging effects. I don't have the backgroung knowledge for this.

In your list of work steps you write under point 5 that you work with cover slip up. For best image quality it is best to have the diatoms sticking directly to a cover slip of the right thickness. I guess that your diatoms might sink down to the slide and only the thickest ones will reach to the cover slip. Have you noticed this effect with your slides? I place my slides with cover slip down onto a screw nut of the right spanner size. To prevent the diatoms from sinking down later I store my slides cover slip down in book sized boxes for 100 slides each.

It looks very much like I have to try your mountant in the near future! Cinnamon Bark oil is easily available here in Germany.

Bob

[einman]

einman
Optical glass adhesives were indeed mentioned. Norland products like Norland 61 and others. They are based on nano zirconia, as far as I know, and that imparts the high RI. Hair sprays sound interesting! However, chemicals that boast a high RI are mostly hazardous, like bromo-naphthalene, they can be polyaromatic and toxic - I doubt that such substances will be allowed to touch the skin of hair.

As a Cosmetic Chemist I can say there are silicones used in hair applications having a high refractive index. Google Silshine 151. It is sold into both the hair and cosmetic market and touches the skin.There are others as well.

I am the inventor on several us patents for hair formulations incorporating silicones so I can say with confidence safety is not an issue. However, given these materials do not "dry" I am not sure how effective they would be as a mounting agent. The specimen could potentially migrate beneath the cover-slip. Compatibility with other solvents would be questionable. Silicones in general are hydrophobic so compatibility with specimens containing water would be something else to be considered. There are also silicone gels with RI of 1.46 and higher. They would reduce/eliminate the migration issue but again not sure compatibility with solvents used in clearing etc. The fact they are not routinely used would suggest incompatibilities or issues. It is something I intend to explore this year.

[Hobbyst46]

Micro-Bob

About the RIs of the components, yes: I thought of the mixture after I noticed the relatively high RIs of the components. Actually it was CBO that attracted my eye, then I thought to add PS. I believed from the start that the resulting RI of the mixture would be some average of the two, roughly 1.6, but I have yet to demonstrate it at least to myself.

I have just discovered an old Zeiss Refractometer, a totally mechanical one, the type that used to be connected to a block heat controller (to supply water at 20 deg celsius, since the RI is really nD20!) and an external sodium lamp. If I can hook up a yellow LED source and FIND THE INSTRUCTION MANUAL of the instrument, I can perhaps measure the RI. Otherwise I may ask you to find a more detailed description of Mr. Rosenfeldts methods.

I like very much your input about the potential benefit of this mountant for diatoms that do not need an RI>1.6 to be clearly seen. My own selection of diatoms includes a few species collected from a pond; I prefer to try and collect more, rather than buy diatoms from around the globe.

Equally, or even more important, is your suggestion about the orientation of the slide during preparation. Indeed, my diatoms might have sank from the cover slip onto the slide itself, because the slide was in contact with the hot-plate, for efficient heat transfer. I cannot be sure, since mostly the small diatoms - say, less than 10microns - appear to be deep into the mountant and far away from the cover slip. Yet, your technique is very probably better!! I will find a small disk of brass (my cover slips are 22mm round), place the cover slip on it and add the slide on top! I should have conceived this before, but did not...

A screw nut is probably fine, especially if it is made of brass and not steel. Coming to think of it, a washer may be even better - and they are usually thinner than nuts!
Thanks.

[Hobbyst46]

einman -

Thanks for the info. I found the Silshine 151 and now I understand its name, and its RI is accordingly ~1.51... Although some web sites try to hide it and the other physical properties. Perhaps there are hair sprays of even higher RI. I was really looking for the highest RI of readily available materials.

I am not sure by how much does the PS-CBO harden. But, after reading some papers about mounting in resins, I thought that a plasticizer may be benefical for mounting, to prevent the mountant from becoming a brittle stiff film. Some mountants include a plasticizer. The various natural components of CBO serve, I hope, the double purpose of solvent and plasticizer. Their vapor pressures are not high, although they are higher than those of phtalate esters for examples. But phtalate esters are harmful and have been phased out from chemical products, as far as I know.

In addition, the PS-CBO is probably soluble in acetone and some other organic solvents. PS itself is quite resistant against the penetration of water vapor. So I prefer to seal the slide with something, like gel nail polish, that will minimize contact with air humidity.

Having started to read the review in www.mapress.com mentioned by MicroBob, I am really surprised by the enormous effort and diversity of formulations by microscopists for more than a century to create mounting media that will last for 50 years. Walter Dioni reviewed "only" about 20 formulations...

[MichaelG.]

einman -

Thanks for the info. I found the Silshine 151 and now I understand its name, and its RI is accordingly ~1.51... Although some web sites try to hide it and the other physical properties.

Yes it does look promising ...
https://www.momentive.com/en-us/product ... -bulletin/

MichaelG.

[MicroBob]

There is a second document on measuring refractive indices: http://www.mikrohamburg.de/Tips/T_Brechungsindizes.html
Pictures are in separate links in the document. Maybe this helps.

For a manual of the Refraktometer you might look here: http://www.mikroskop-online.de/Zeiss%20Jena%20BDA.htm

It really is impressive to see the vast amount of microscope techniques that have been developed mainly in the last 150 years. Some of them have probably not been revised in the last couple of decades. Since then new materials have been invented or are at least better available so it is very well possible to find something new today. On the other hand side many materials have disappeared or are difficult to obtain today. I also have the impression that many old recipes didn't care much about practibility in the home laboratory and safety and environmental aspects. Therefore alternatives that offer 80% performance with 20% hassle are very welcome.

I recently tried LOCA, the UV hardening stuff to glue glas screens on smartphone displays, as a mountant for radiolaria. The results were very good. It' possible to make ready to go radiolaria slides within a couple of minutes with this mountant.

[Hobbyst46]

Microbob

The second link brought me indeed a manual for the Zeiss refractometer. Although it refers to a more advanced version than the vintage instrument that I can use, most of the instructions must be similar. Thanks a lot.
It would be intersting to know the details of the LOCA protocol - which brand, how to apply it, curing etc. Does it need a specific UV source or just good old direct sunlight at noon.

[Micro-Bob]

I opened a new topic on the LOCA mount for Radiolaria to avoid mixing things up: viewtopic.php?f=10&t=5565

Bob

[Hobbyst46]

Supplementary information, about 3 months after the mounting syrup was conceived.

1. Refractive index - measured with a very old but excellent Zeiss refractometer, with a 593nm (+/-) LED light at a temperature of 22-23 (the standard is 20deg C). Water is 1.331, a certain Olive oil was 1.467, CBO was 1.551, and PS-CBO was 1.567. Since the RI of PS is 1.6, the result is reasonable. Naturally the RI can vary, since CBO is a natural product.

2. During preparation, it is important that heating and the weight on the slide do not move relative to each other. Furthermore, Micro-bob's advice, to place the slide with coverslip face down on the heating plate, i.e. the coverslip touches the heating surface, was adopted. A Slide heating frame was constructed from scrap aluminum, using hand tools (shown). The slides are placed within bounds, under weights, and the frame is placed as a cover of a pot of boiling water (instead of a hot-plate...).

3. Gel nail polish is a useful sealant in this case since it is more resistant to CBO than ordinary nail polish. I cure gel nail polish with sunlight or with a home black-light 60cm fluorescent tube (favored by teenagers to create "atmosphere"), 15 minutes and it becomes hard, though slightly elastic.

4. Provided that just a tiny drop of PS-CBO is applied for mounting, such that it does not spread beyond the coverslip, the slide remains useful for 3 months, optically clear, no yellowing, no drying out or crystallization. The mountant does not harden to a brittle mass, presumably because the liquid constituents of CBO serve as plasticizers.

5. Altogether, the PS-CBO has a potential, in my opinion, as a relatively safe and accessible medium for STREW slides, at least for several months.

I thank again all members who commented on this post, and whose comments were always helpful and informative.

The diatom photos are still a novice work - please ignore the background...
Were taken with a 63x/1.4 PlanApo oil immersion, Canon M10 camera with the kit zoom lens at 45mm, placed over the Zeiss 10x eyepiece in the phototube (ordered adapters are in transit).
The brightfield is a stack (Helicon B) of 5 photos 2um apart each,
the phase is a single frame under green filter.

[MicroBob]

The diatom looks a bit smashed. Maybe you could reduce the pressure on the slide a bit. When you make your slide upside down the distance between cover slip and slide in not so important any more. The diatoms are always where you want them, regardless of how much mountant is below. Your fixture is nice!

Your short time experiences and measured refractive indices show that you have invented a useful mountant! The refractive index might climb a bit with time and evapouration of volatile components. Even if it would go pitch black after 4 month there would be uses for it. With its refractive index it is not among the "top mountants" of the old diatomists, so it is perfectly feasible that it is just fine in all other aspects and nobody has had a closer look in this direction so far. The classic "top diatom mountants" like Naphrax, Pleurax and Aroclor require knowledge and absolutely a laboratory vent to make, making naphrax is time consuming (working and sleeping in shifts...), and Aroclor is not allowed to make for decades and the recipe in not publicly known. I know people who cooked mountants, but even they don't make these mountants on anything like a regular basis. Todax the pinnacle of diatom work is the SEM anyway. So for the hobby diatomist without connection to a microscopy group with resources it is great to have something available to get started without many compromises. It would be really useful for the micscopy world if you carry on you with your work with this mountant and give an update on the behaviour of the slides now and then.

Bob

[Hobbyst46]

MicroBob-

Indeed, I have noticed that many of the diatoms I collected and cleaned, especially the long pennates, are broken, so maybe I should use lighter weights or omit them as you suggest. I thought once of picking out the unbroken ones and making arranged slides, but gave it up.

I do follow the status of the slides and will update.

Thanks.