Comparison of mountants for diatoms

[Radazz]

This is a spin-off of the discussion about using UV hardening glue for mounting transparent samples.

I bought a small kit of Soft, Thick and Thin UV hardening lacquer for making trout flies.
I wanted to try a mounting Media that was the consistency of Canada Balsam, but was easier to find and would dry hard enough to hang upside down in the inverted microscope in less than a week. I used the thin variety which flows like diluted Canada balsam.

I had purchased a small sample of cleaned diatoms from Diatoms.com in the Netherlands.
These are small samples, and I didn't want to waste them learning how to mount them, so I did some experiments with diatomacious earth.
Once I found the proper consistency to get a strew that was not so choked with diatoms as to be useless, I started working with my precious little sample.
What worked for me was to place the sample on a coverslip. I use a slightly cloudy drop but no thick chunks. I let this air dry, and then drip the media onto the slide surface and drop the coverslip with the dried sample face down onto the mounting media. A light tap to clear any bubbles, but be careful not to slide the coverslip around on the slide as this will damage the diatoms. That fantasy of getting a perfect slide on the first try will be dashed, but you will have fun working out the bugs.

Here is a comparison of three different materials:
UV glue, Clear nail polish, and Canada Balsam diluted with xylene. (Please take necessary safety precautions handling this type of preparation)
These are all one shot, no stacking. The important thing is the mounting medium, right?

Olympus IX70, 20x objective, Canon EOS 7D
First is 2 shots of a slide I mounted with UV glue:
First in HMC

And in Brightfield


These next two are mounted in clear nail polish
HMC

Brightfield


And finally a pair mounted in Canada Balsam
HMC

Brightfield


I see some real differences in HMC, I couldn't tell you the refractive index of any of them.
The major problem with the UV cement is bubbles. Will have to try putting the slide in a vacuum chamber while it is still liquid, then into the ultraviolet light.

I don't believe i'm ready to chuck the method by a long shot.
The mounts of insect parts I made were too thick for the thin glue, but the thick variety builds up nicely around the specimen and gets a bead around the cover-slip not unlike a Carolina slide.
Curing a deep mount in UV can get shadows around the specimen if you don't get coverage all around.

Also not a great idea to set the slide on top of a UV flashlight with multiple LEDs as they burn a pattern into the mount which is invisible in brightfield but can be seen faintly in phase contrast or HMC. They show up well if you shine a UV flashlight on them.

Personally I prefer the Nail Polish for brightfield, and Canada Balsam for HMC, but stacking just might change that opinion.

Enjoy,
Radazz

[Radazz]

Later today or tomorrow I will do Phase Contrast

[Hobbyst46]

Thank you very much for this methodical comparison!
I am especially impressed by the HMC, it yields good contrast in all mounting media. I agree that the Canada Balsam looks nicest.
The best BF IMO is in the UV-curable glue. Perhaps its refractive index is significantly higher than 1.5. However, I am not sure whether the frustules are not filled with air, and the difference between the RI's of air and the medium enhances the visibility of the diatoms. Some of the frustules appear as if they have been only partially filled with medium, and air is trapped inside.
My own experience is that practically all viscous media are prone to air bubbles...although in your demonstration, the Canada Balsam seems to be free of them.
Eagerly waiting to see the phase images!

[photomicro]

I am a little confused at this, being unsure of the intentions.

For diatom frustules that have been cleaned, being siliceous, they have a refractive index (RI) comparable with glass. Hence to mount in a mountain of similar RI would render many details very feint. You need a material with a higher (or lower, hence even cleaned diatoms show up well when mounted dry <RI=1> , or in water <RI=1.33>) RI.

This is why most diatom specific mountants have an RI between say 1.65 and 1.80.

Now, a few caveats;

1. This applies for brightfield, but if you are going to use phase, DIC, HMC or other contrast enhancement techniques, then you will get contrast and hence visibility with any mountant.

2. It is important that a mountant gets into the frustule, bonds with the glass, and helpfully doesn't form bubbles. Longevity is an issue too, if the slides are to last. I have slides well over 100 years old that are fine.

3. Large frustules like the Pinnularia shown in the photos are thick enough to show up in most mountants, though the details of striae will not. Thus, for ease of use, I have mounted Arachnoidiscus in crystal-clear balsam substitutes such as Eukitt.

[Hobbyst46]

I couldn't tell you the refractive index of any of them.

The RI of Canada Balsam is very similar to that of most glasses, around 1.52. The RI of common nail polish (made of nitrocellulose mostly) is very probably around 1.5. The RI of the glue is possibly higher than that, but not very much - just my guess, 1.52-1.55.

[Radazz]

Here is the Phase Contrast comparison:
Olympus BX40 20X Ph Canon EOS 7D

Canada Balsam


Clear nail polish


And UV Glue


Really comparing the clarity of the mounts. Also testing the camera mounts on both scopes.

Radeazz

[Hobbyst46]

Thanks again for posting these data!

Consistently, with each medium, the phase contrast looks better than the BF contrast - as expected.
And, as photomicro commented above, phase contrast and HMC improve relative to BF, with any mounting medium.
Personally, I favor the Canada Balsam for the HMC, the glue for phase. The nail polish images come last.

[Radazz]

Reading back through this thread, I realized I hadn’t tried dry mounting them.
I grow my POL crystals on the coverslip as well, and have had some very good results dry mounting them. The crystals are never damaged by the mountant.

Definitely going to give that a try.

[MicroBob]

Hi Radazz,
it looks as if you had Coolpix-Rings! (The rings in the right half of the images) This name comes from the formerly very popular microscope camera adaptation of an old Nikon Coolpix 995 to a Leitz Periplan eyepiece. Some plastic lenses in the Nikon Objective produced these rings when used with strong objectives.
What optics do you use between Microscope objective and camera sensor?

Bob

[Radazz]

Looks like dry mount is the way to go.
Olympus IX70 20x HMC 2 shot stack, picolay

Radazz