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

[Hobbyst46]

For diatoms, a high refractive index (RI) mounting medium is wanted. Commercial resins or solutions of RI 1.6-1.7+ are expensive, or hard-to-get, or have a limited shelf-life, or contain toxic solvents like toluene. Looking for a more "domestic" stuff, I conceived a (apparently new!) mounting formulation- PS-CBO: a solution of stiff white styrofoam (SF) in cinnamon bark oil (CBO).
CBO is a freely available food and cosmetics ingredient. It is a yellowish liquid mixture of cinnamaldehyde (50-70%) and other essential oils. The RI is 1.55. Literature indicates that CBO has occasionally been used for mounting diatoms, but in my experience the specimen deteriorates within days, at least because CBO diffuses through the nail polish and evaporates.
I may have found that a viscous solution of (ca. 10%) polystyrene (PS) in CBO can be used for semi-permanent (at least) diatom mounting.
PS has an RI of 1.6. White stiff styrofoam (SF), the ubiquitous packing material and heat isolator polystyrene, is actually PS. One commercial mountant (DPX) contains PS, a solvent and a plastisizer, however, its RI is only 1.52.
Notice: CBO is odorous and might be an irritant to the skin, eyes and throat. I keep it in a hermetically sealed bottle, handle it with latex or rubber gloves and use it in a ventilated space.
To make PS-CBO, start with 10ml of CBO in a 30ml (say) hermetically-sealed glass vial (plastic seal, not rubber/ silicon-rubber). Cut an SF board into 1x1x1cm pieces (its density is 0.05g/ccm). Push as many pieces as possible into the liquid (use a toothpick). Stopper the vial and shake a few times ("shaken- not stirred!"...). Wait a few hours until the SF disappears. Repeat SF addition and shaking 10-20 times, the aim is a very viscous solution. Filter it, if necessary, through a 0.45micron syringe filter into a dark-colored, hermetically sealed storage vial.
To mount diatoms, (strew slide) let a drop of aqueous suspension of the diatoms dry out on a 22mm coverslip. Place a small drop of PS-CBO on a slide. Invert the coverslip onto the drop. Place a 50g weight on the coverslip. Seal with nail polish, or, for best results, heat the slide at around 100C for 10min prior to sealing. Heating transforms the mountant into a slightly sticky, semi-hard layer. I believe that its RI is 1.55-1.6. It is not strongly bonded to the glass - can be mechanically peeled away with a sharp tool.
Here are my preliminary strew slides of a sample of diverse fresh water diatoms from a pond. They appear to be transparent, colorless and bubble-free after a couple of weeks. Time will show if they are indeed "semi-permanent". I will probably attempt to optimize the heating.
The photos below were taken with a hand-held Canon Powershot 720 camera on top of the Olympus eyepiece on a Zeiss GFL, brightfield, 16X/0.32 Plan or 40X/0.75, 6500K led light with a KR12 filter, automatic white balance, cropped to fit file size limits.

[zzffnn]

Hobbyst46,

Nice work, thank you for sharing.

I assume you do not have a refractometer? If the medium's RI is 1.55-1.6, then it may be useful for most diatomists, except for those who do dotting on Frustulia rhomboides or Amphipleura pellucida.

Uncontrolled random strews without comparison may not provide solid conclusion though. Maybe get some loose/cleaned Gyrosigma balticum or Pleurosigma angulatum, and use those resolution targets to compare media? I am guessing Klaus Kemp may be able to sell those, though an US source would be far cheaper (sorry, I don't know such a source - would like to find one though).

This glass glue may work better than nail polish as sealant: https://www.hobbylobby.com/Crafts-Hobbi ... nd/p/28110

Or it may even be used as mounting medium. It dries quickly by itself to a glass like clarity, binds well and does not seem to shrink much (I am running an experiment to confirm shrinkage or lack of). I don't know its RI or key components, though it does contain (the harmful) xylene as solvent.

Hobbyst46, would you be wiling to test that glass glue, if it does not shrink much (I can gift you a tube)?

[Hobbyst46]

Hobbyst46,

Nice work, thank you for sharing.

I assume you do not have a refractometer? If the medium's RI is 1.55-1.6, then it may be useful for most diatomists, except for those who do dotting on Frustulia rhomboides or Amphipleura pellucida.

Uncontrolled random strews without comparison may not provide solid conclusion though. Maybe get some loose/cleaned Gyrosigma balticum or Pleurosigma angulatum, and use those resolution targets to compare media? I am guessing Klaus Kemp may be able to sell those, though an US source would be far cheaper (sorry, I don't know such a source - would like to find one though).

This glass glue may work better than nail polish as sealant: https://www.hobbylobby.com/Crafts-Hobbi ... nd/p/28110

Or it may even be used as mounting medium. It dries quickly by itself to a glass like clarity, binds well and does not seem to shrink much (I am running an experiment to confirm shrinkage or lack of). I don't know its RI or key components, though it does contain (the harmful) xylene as solvent.

Hobbyst46, would you be wiling to test that glass glue, if it does not shrink much (I can gift you a tube)?

**************
zzffnn - thanks for the response.
I do not have a refractometer. Cheap refractometers for wineries and breweries only go up to RI=1.3. Research grade refractometers are expensive. Moreover, I do not know how to measure the RI of a film of polymer or mixture between two glass layers. I hope to find a way around this. In the meanwhile, I assume that the RI of the mixture is somewhere between the RIs of the components, so 1.55-1.6. These data are from literature and I hope to verify my guess.

Although I am no beginner in microscopy, I am an amateur and beginner at both diatoms and photomicrography. This are my first ever sample of diatoms. I cleaned them with bleach and HCl, and I admit the result is unappealing, but better cleaning and separation are my future plans. I am not a USA resident, so postage of samples will be costly anyway. I would very much welcome info about who on earth sells diatom specimens for mounting, at reasonable prices.

Regarding the glass adhesive, I can look for it in hardware or crafts and hobby stores. But I doubt that it will have a high RI. The high RI of stuff like Naphrax and other known media is not due to the solvent, xylene or toluene or benzene or alcohol. But I will have to check the properties of constituents mentioned on the package in the literature and try to reach a conclusion.

There are certain Norland (USA) adhesives, however, that are UV- or VISIBLE light curable. They are clear liquids that solidify and bond glass when illuminated. They are Norland 61 and some other adhesives. Are listed on their site, with tables that tell you their physical properties and RIs. Some have RI up to 1.7. This is achieved by high-tech - I think, nano crystals of zirconia, again a material with high RI. Why I dont use them - cost, shelf life etc. But I am sure that they are fine for diatoms.

I fully agree that my photos are quite poor quality. But to my eye on the microscope itself, the specimens look much better. I compared the contrast and resolution of the diatoms in PS-CBO to those of diatoms in 77% fructose, and I think there is significant difference. In my defence, I can only suggest that these are preliminaries. I do need to get a decent stable camera and learn how to take pictures that will do justice to the views through the microscope...

[Hobbyst46]

zzffnn,

One thought that crossed my mind is how is any adhesive fit for mounting. Supposing that the diatoms are lying flat on the slide. If you heat the slide to such high temerature that they partially melt and "welded" to the slide - forming a "glass bridge" - then after cooling you can use any adhesive as mountant. But if you only have the diatoms resting on the slide, pre-arranged or at random, and you add an adhesive (apart from EPOXY!), what will prevent the diatoms from bonding wildly by the stuff and spread around the glass in an unpredictable manner?

From what I have read, the trick is to first spread a very thin layer of adhesive on the slide, then arrange the diatoms, then mount. Is this how you intend to use the glass bond?

Hobbyst46

[zzffnn]

Hobbyst46,

Sorry for the confusion. I did not call your image quality poor. They are typical, considering a strew mount and hand held camera. I got similar results before, with a similar set-up.

I don't doubt you, if you have done comparison. I was just saying, rather than using random diatoms, using well established test diatoms would provide better idea. Sourcing specific loose diatoms may be a problem though.

You are probably right about the glass glue not having high enough RI. If high RI is not easily/cheaply achieved, then there is no point of making/selling glass glue with RI of 1.6-1.7. After all, cheaper type of glass, which the glue is supposed to bind, usually has RI of less than 1.55.

I mentioned xylene, because it is a somewhat harmful solvent and you paid attention to selecting safe reagents.

You made a great point about how to use adhesive as mountant. We surely don't want diatoms to move too much with the mountant.

[MichaelBrock]

In the Diatom Forum over on yahoo groups someone started a thread earlier this month (10/6) saying that they were experimenting with polystyrene dissolved in toluene. The problem they ran into was that it required so much toluene that when they were drying the mount on a hot plate the mountant shrank and left gaps. Here is the thread but you'll probably need to sign up as a member there first to see it:

https://groups.yahoo.com/neo/groups/dia ... opics/3864


Michael

[zzffnn]

Hobbyst46,

The following 3 web sites are established diatom arrangers/sellers. They are all located in Europe and may be able to provide you loose (unmounted) test diatoms.

https://www.diatoms.nl/index.php
http://www.diatomshop.com/product-list.html
http://www.diatoms.co.uk

I live in US but don't know any US diatom seller
Anyone knows? Maybe Charles can start such a business

[Hobbyst46]

zzffnn - I would really post better photos if I could.

I have carefully read your posts about the glass bond from HOBBY LOBBY in other your threads. Looks like I should try it or a similar glass bond from crafts stores, at least for sealing around the coverslip, instead of nail polish. Who knows, it might be a better sealant and bond material than nail polish.
And if I find access to a refractometer I will check the RI as well. And post the results...

[Hobbyst46]

zzffnn - thanks for the links.

Michael - Thanks for the post. I did not read the link about polystyrene and toluene. They possibly removed all of the toluene, without due press on the coverslip, so the solvent vapors escaped too rapidly for the PS residue to establish itself in the space between the glass layers.
The DPX medium contains a plasticizer and I do not know if one is supposed to heat to remove the solvent.
My approach is different: The CBO is not that volatile and I did not attempt to remove it altogether. Rather, I aim towards a very viscous resin-like of the CBO-PS mixture. Hope that it will preserve this form for a long time. Being sealed. Perhaps with something better than nail polish.
I doubt if it can be heated to much above 100 degrees C, since then PS undergoes physical transitions that might alter the film properties.
Bottom line: "It is experimental!"...

[Hobbyst46]

P.S.: I somehow forgot to mention that when mounting diatoms, the drop of PS-CBO should be small, such that spreading under the weight on top of the coverslip will still leave some space under the coverslip. So, the medium will not be in direct contact with nail polish. This is because, as I mentioned before, CBO penetrates through nail polish.

[MicroBob]

Hi Hobbyst46,

thank you for posting the results of your work! If you find a nice and simple diatom mounting medium, many people will be very happy about it.

I don't think that you have to buy diatom material at this point to test the properties of your new mounting medium. From my experience diatoms cleaned with bleach look really good in the usual mounting medias.

Your pictures show that the diatoms are not lying flat on the back of the cover glass in one thin layer as it would be necessary to get the best image.
I would reduce the density of the strew and try to keep the diatoms at the cover glass as goog as possible. It helps to make the slide upside down (cover glass at the bottom) and also always store it that way. Your pictures would instantly become a lot better.

Here the measuring of the refractive index of mounting media is described in german:

http://www.mikrohamburg.de/Tips/T_Brechungsindizes.html

The production of Pleurax is not too difficult but you absolutely need a laboratory fume cupboard (Not in the garden with the wind from behind ):

http://www.mikrohamburg.de/Tips/T_Hochb ... ittel.html

[photomicro]

I would recommend Pleurax. It has a high enough RI, and is usually in alcohol, giving a number of benefits;

1. Not as toxic
2. Not as sensitive to any water in the prep
3. Can indeed be used with uncleaned diatoms, preserving the innards. Obviously striae detail is absent but it allows connected forms such as Tabellaria to maintain this.

If you are serious about trying different mountains, I could supply some, as a friend made a batch a couple of years ago.

I am in the UK.

Micr

[Hobbyst46]

Hi MicroBob - thanks for the post and for the very important remarks.

I already attempted to lower the density of the diatoms on the slide, to obtain a "monolayer". However, your point, that a less dense layer will also improve the photo quality is great!!

And many thanks for the link about how to measure the RI without a dedicated commercial refractometer! These methods I had vaguely in mind, but did not know how to make them work! I am very happy with these protocols, and hope to try method 2 in the protocol from the link.

Regarding Pleurax and other synthetic resins, the syntheses described by G Rosenfeldt and others are indeed fairly straightforward - in the hands of trained organic chemists, and the facilities of well-equipped laboratory. My ambition was only a recipe for a mountant that can be easily prepared by amateurs at the back yard.

The problem that I observed was that the CBO-PS mountant penetrates through nail polish, due to mutual solubility. "Gel" nail polish, curable by UV irradiation or even sun-light, may perhaps be better, since it is more resistant to acetone, for example. My experiments are now focused on this.

And, needless to say, CBO-PS is not yet mature for arranged diatom slides.

[MicroBob]

The usual microscope objectives are calculated to find the object directly below the 0,17mm cover glass. Even when you manage to focus much deeper the image quality is not up to specification due to this additional layer of mountant. You can just dilute your diatom-water-mix further to get a strew with less density. Perfeclty cleaned cover glasses allow the drop to spread well. Let the drop dry without moving the coverglass, otherwise the diatoms tend to clutter together.

You can use shellac to stick diatoms to the cover glass. Make a saturated solution of shellac leaves in ethanol, then make a 1:20 dilution of it and put a half drop on the very clean cover glass so that it spreads out well. When your diatoms are in water just put a drop on the dried shellac, let i dry out and heat the shellac to 90 ° C. The diatoms will stick to the cover glass.

I don't have access to chemical laboratory, so a real simple mountant would be good for me too.
You might try wax or silicone to seal your slides.

[Hobbyst46]

Thanks MicroBob for the additional info and the idea of shellac glue for the diatoms. I will check where I can get shellac flakes.

Silicon glues contain organic solvents and are probably attacked by essential oil, so I think that for sealing, I prefer gel nail polish. If that fails on the long-term , will try waxes. The wax I know is smelly when melted...

[MicroBob]

A sealant for motor casings might work also, it should resist oil.
Shellac is used by artists, and craftsmen, e.g. for veneering and for gold-plating.
Where in the world do you live?

[photomicro]

Regarding Pleurax and other synthetic resins, the syntheses described by G Rosenfeldt and others are indeed fairly straightforward - in the hands of trained organic chemists, and the facilities of well-equipped laboratory. My ambition was only a recipe for a mountant that can be easily prepared by amateurs at the back yard.

When you consider how little mountant you actually use, a 30ml bottle will last a long time, so I don't see why we all need to be able to make our own. At least the proven mountants are known to have longevity and penetrate into the diatoms. If you find something that works, stick to it I say, you don't to find your slides are useless a few years down the line.

Don't get me wrong, I have experimented, and it is fun, but for slides I want to be good, and last, I use one of the well-known ones.

[Hobbyst46]

I live in Israel.

MicroBob - thanks for the tip. I will look for shellac in crafts stores and pharmacies. In hardware stores it is only sold as a solution in methylated spirit or perhaps turpentine. So I will look for flakes. High-temperature silicon sealant for engines and ovens is indeed a possibility ifvit is not to viscous.

Photomicro - like you say, it is fun. And curiosity. These are the triggers.

[mrsonchus]

Hi hobbyist46, I've followed with interest your adventure here with the formulation of a good diatom mounting-medium. As photmicro says, it's very easy and economical to simply purchase a very good, tried-and-proven mountant 'off the shelf' - but, where's the fun?!
I completely agree with you re the sheer fun and stimulation that the tiniest little question generated by our brains may prove to be that spark of intellectual interest that will occupy and delight for so long!

I've no experience in this area at all - I'm not 'of the frustule' - but I've been following along with your every post and response with enjoyment and interest - thanks for this unusual and interesting contribution.

Keep up the good work, remember, even if ultimately you may use the established mountant/s for your diatoms, as photomicro says, you'll have in no way at all failed. The experimental process and an enquiring-mind are after all a 'marriage made in Heaven'!

Enjoying your adventure and learning lots too - keep it up to whatever point suits you my friend - it's all new knowledge and a stack of fun along the way!

[Hobbyst46]

Thanks, John B, for the support. It does stimulate me a lot. And it is a real pleasure to improve one's methods on the basis of consulting with experts.

[mrsonchus]

Thanks, John B, for the support. It does stimulate me a lot. And it is a real pleasure to improve one's methods on the basis of consulting with experts.

Bang-on my friend - and this forum's full of real experts in this field, and as a bonus - they're an extremely kind, friendly and helpful bunch!

[Crater Eddie]

This glass glue may work better than nail polish as sealant: https://www.hobbylobby.com/Crafts-Hobbi ... nd/p/28110

I looked for this in our local Hobby Lobby today and didn't find it. What section is this in? I did look by the beads.
CE

[zzffnn]

CE,

It was in a glue section between the glass crafting aisle and the beads aisle, in my local one. But another Hobby Lobby that I went to arranged sections/aisles differently.

Better ask your local store assistants (show them the product photo from the web page), especially those working in nearby aisles. Their cashiers do not have an electronic database to help you locate a particular item, unlike HomeDepot.

[MichaelG.]

This glass glue may work better than nail polish as sealant: https://www.hobbylobby.com/Crafts-Hobbi ... nd/p/28110

Or it may even be used as mounting medium. It dries quickly by itself to a glass like clarity ...

.

That looks a useful material, at a very good price [thanks for mentioning it]
Unfortunately I have not found Hammerhead products for sale in the U.K.
Our "glass-bond" adhesives seem mostly to be UV-curing cyano-acrylates.

My first guess was that it might be a Silicone RTV sealant/adhesive [such as that used for assembling fish-tanks] but, given your mention of Xylene, I am not confident ... I would be interested to see its MSDS if you can locate a copy.

MichaelG.

[zzffnn]

Yes, its texture reminds me of silicone. But unfortunately, I lost the original package. If it is not online, we may have to wait for CE or other members to get theirs.

[Crater Eddie]

It will be next weekend before I get back to the town with Hobby Lobby. The odd thing is that I can't find any useful information on this glue online... no data sheet, no MSDS, nothing. Amazon has a listing for it but says it is currently unavailable. Odd. Oh well.
CE

[MichaelBrock]

I don't know anything about the hammerhead brand or if there is something special about it but Loctite also makes a glue intended for glass that they market as "crystal clear and dishwasher safe". It's Loctite 233841. Amazon and some of the box stores also carry it. I'ts on my agenda to test it should I find some diatoms needing mounting.

Edited to add: there are also quite a few inexpensive optical cements/glue/adhesives sold on ebay from China. I know of at least one microscopy person that has used these to repair compound lenses. They appear to be UV cured:

https://www.ebay.com/sch/i.html?_from=R ... e&_sacat=0

[Hobbyst46]

High All,
One advantage, in my opinion, of UV curable gel nail polish over UV curable glass cements, at least for sealing, is that the former is easily spread around the cover slip with its dedicated brush. Glass cements that quickly harden may be too fast-acting for this application.
Also, the nail polish keeps very well in the bottle. A long shelf life.
For mounting, a known commercial UV-curable glass cement with a high RI is the expensive Norland 61.

[Hobbyst46]

Happy New Year everyone,

My original idea was to formulate from readily available and non-toxic substances a home-made, medium-high refractive index mounting medium for diatoms. Not to compete with Hirax, Zrax, Naphrax, Pleurax or Norland 61, but to enjoy the hopefully new formulation.

Three months after, with more experience, having prepared more slides, here is the slightly modified protocol for a STREW slide:
1. Clean the diatoms - there are many methods, the choice depends also on the composition of the collected raw sample. My sample was taken from a tiny ornamental fish pond. Cleaned it with bleach, then HCl, then H2O2 and boiling.
2. Let a drop of the suspension dry completely, say in 48 h, on a cover-slip - like in other methods of diatom mounting.
3. Place a drop of CBO (Cinnamon Bark Oil) on the dried stuff, and blot most of it with a filter paper (touch it gently). This is to facilitate the removal of air from the inside of the frustules. I believe it works.
4. Place a small drop of the PS (polystyrene)-CBO syrup on a slide, invert the slide onto the coverslip.
5. On a hot plate or boiler plate at ca. 82-85 degrees celsius (see below) place the specimen, coverslip side up, and heat for ca. 15 min. I put a small weight (a 1/4" brass tube fitting, weight ~40g) on the coverslip. Do it carefully, use forceps, and by all means avoid having the coverslip slide to and fro under these manipulations.
6. After cooling, seal with gel nail polish ("top gel"), if you can cure it with either UV irradiation (3-4min) or good old direct sunshine (1-2h at least). Otherwise, seal with ordinary nail polish.

In my hands, the mountant becomes very viscous, nearly hard, not quite as hard as resins, but fairly stable under slight pressure. Mounted diatoms do not move. No air bubbles appear. No discoloration (although CBO itself is yellowish). Gel nail polish is more resistant against CBO than is ordinary nail polish, and is a better sealant in this case.

In all, the prepared slide appears to be stable enough for visual inspection even after at least 3 months, although I did not attempt to verify that the location of each diatom on the slide does not change with time. My mechanical stage is not sufficiently reproducible for such tests.

Neither did I measure the RI of the syrup. Attempts to apply various improvised "refractometers" failed, because I could not focus on the home-made optical edges. I must find a commercial refractometer for that...

Boiler plate: use a small stove cooking pot, e. g. a "Turkish Coffee Pot" that has a flat, horizontal top. Cover it with a flat aluminum plate, of sufficient area to cover the pot completely and leave a narrow slit for the vapors to escape. Plate thickness about 2 mm. When water is boiling inside, the temperature of the plate will be 82-85 Celsius. When room temperature is about 20-25 Celsius.

Show below are some diatoms (unidentified by me...) in plain phase contrast views. Zeiss GFL. Plan-Neofluar 40x dry or Planapo 63x immersion objectives. Green bandpath filter. Smartphone camera on a modified adapter enclosing the eyepiece (like in the other post). Open Camera software application, camera zoom 2x. Single photos, resized (software - Irfanview. One of the greatest freeware tool for image handling IMHO).

[Micro-Bob]

Hello Hobbyst46,

here: http://www.mikrohamburg.de/Tips/DER%20B ... SINDEX.pdf you can find a method for determining refractive indices. It's written in german but you probably can use googel translator on it. For unknown reason its the most downloaded document of our site.
For a practical test you could just compare CBO with a resin of known refractive index to get an idea where you are round about.

Bob