diatoms

[frank/mic]

Hi
I have recently re-registered after a prolonged absence and, to be honest, never really getting to grips with the technology. So my question is....why do diatoms need such a specialised ( and expensive ) mounting medium as Naphrax. I have read somewhere that a usable alternative can be made by dissolving polystyrene in cinnamon bark oil and have dissolved it in nail polish remover but not as yet used it. Any advice / comments would be welcome.
Cheers
frank

[Wes]

As I understand it you want a good difference between the refractive index of the sample (diatoms in your case) and the surrounding medium. These differences cause a phase shift (invisible to human eye) between the light fronts passing through the sample and the ones surrounding it. The phase shift is then converted to an amplitude difference (dark or bright) by techniques such as phase contrast and DIC.

Because diatom frustules are made out of silica this has the same refractive index as glass (which in short is also silica) and common mounting mediums like Canada balsam you won't get much of a phase shift with standard mounting media and therefore no contrast.

Naphrax and Zrax have a refractive index of about 1.7ish whereas silica is around 1.51.

I also though about polystyrene as it resembles Naphrax and Zrax structurally (a phenyl-rich polymer) but never found anything about its use in diatom mounting.

[Hobbyst46]

Naphrax and Pleurax are good but are not cheap, especially considering shipping costs, import taxes etc.

Cinnamon Bark Oil (CBA) has an extremely high RI (for an oil!), of about 1.55-1.58 or so. Not as high as Naphrax or Pleurax.
Polystyrene also has an RI of about the same figure. Styrofoam is essentially polystyrene.
Since polystyrene readily dissolves in CBA, the solution has roughly a weighted average RI, so again, 1.55-1.58.
About three years ago I prepared this mixture as a planned home-made mounting medium for diatoms. I thought it might serve as semi-permanent medium. Or, with luck, permanent.
The preparation is easy and does not require any equipment.

It works very well, in the sense that it provides ample contrast for the diatoms, in brightfield as well as phase contrast.
Cons:
1. It is not a resin and does not solidify with time.
2. CBO easily leaks out through ordinary nail polish, and is difficult to seal. The coverslip can only be sealed with gel nail polish, the type that is cured with UV.
3. After about 4 months, an unknown (to me) component of the CBO starts to crystallize out, and ruins the specimen.
4. The amount of styrofoam in the mixture solution should not be too high, otherwise the medium does not penetrate well into some diatoms.

So, I am quite happy with the "invention", it is sort of a semi-permanent mounting medium, no more.
At about the same time, folks in Germany tried a solution of polystyrene in toluene or another solvent, I doubt that they were successful (but do not know for sure).

More recently, I tried NOA61 cement, which has an RI of ~1.56 and is UV-curable.
Cons:
1. Expensive.
2. Fails to permeate into certain diatoms.

For really good permanent slides, choose Pleurax (as recommended to me by Mike, member "photomicro")...

[75RR]

I suppose a syndicate is the way to go if there are enough interested members in one country/zone.

http://www.brunelmicroscopes.co.uk/naphrax.html

As a matter of idle curiosity, what is the cost of the 15ml and 250ml amounts and how many slides would each do?

[MicroBob]

Hi Frank,
diatoms are basically transparent and have a refractive index around 1,45. To get a contrasty image a difference of the refractive index of the mountant of 0,1 is recommended. This doesn't necessarily mean that it has to be higher though. Diatoms in air give lots of contrast but you can't use objective apertures above 1.
Enclose is a picture of a dried out salt water diatom slide, with a bit of DIC tricks.

Bob

[MicroBob]

And this is cleaned diatom material photographed with incident light dark field contrast. When you think of them as made from glass you get the right idea.

[frank/mic]

Thank you so much everyone. I'll potter on and give feedback as and when.

Cheers

Frank

[photomicro]

Hi Frank,
diatoms are basically transparent and have a refractive index around 1,45. To get a contrasty image a difference of the refractive index of the mountant of 0,1 is recommended. This doesn't necessarily mean that it has to be higher though. Diatoms in air give lots of contrast but you can't use objective apertures above 1.
Enclose is a picture of a dried out salt water diatom slide, with a bit of DIC tricks.

Bob

Whilst agreeing to a large extent with this, is in't quite a simple as that, as it depends so much on the diatoms. You could mount nice big specimens of Arachnoidiscus in Canada Balsam or a modern equivalent, and they would show up fine. Do that with Gyrosigma, Pleurosigma and many, many others, and you will see little but an outline. As so many things, it is horses for courses.

This is worth a look;

http://www.microscopy-uk.org.uk/diatomist/ADvol43.pdf

(find page 39, where an article on 'Diatom Visibility Index' starts)

Like you say though, use Phase, D/G, HMC, DIC etc. and things will show up too.

[MicroBob]

Hi Mike,
thank you for the link! This booklet also contains the description of the little field microscope that would be useful for diatom collecting.

Here is a link to a detailed research on diatom refractive indices: http://www.mikroskopie-ph.de/Mikrokosmo ... tomeen.pdf
Apparently the refractive indices depend not only on the species but also on the place they were collected!

I usually make strew slides with Pleurax. For single mounts of certain species it would be an idea to select the optimal mountant of cause. But I'm usually sticking to the Pareto principle: 20% effort for 80% result and thats it!

Bob

[photomicro]

Hi Mike,
thank you for the link! This booklet also contains the description of the little field microscope that would be useful for diatom collecting.

Here is a link to a detailed research on diatom refractive indices: http://www.mikroskopie-ph.de/Mikrokosmo ... tomeen.pdf
Apparently the refractive indices depend not only on the species but also on the place they were collected!

I usually make strew slides with Pleurax. For single mounts of certain species it would be an idea to select the optimal mountant of cause. But I'm usually sticking to the Pareto principle: 20% effort for 80% result and thats it!

Bob

Thanks Bob, my pleasure. Whilst they are only scans, the 'archive' of Amateur Diatomist is a treasure trove of info (*), and the index is searchable.

It was a printed publication, originally supplied free to stimulate interest, but then at (moderate) cost, but printing costs, the lack if authors coming forward, and in the end lack of demand meant it went 'online' and then sort of fizzled out. Disappointing really, when you think of the prices that diatom slides go for on fleabay, sad that more don't try their hand at making them.

I did supply someone on here (or that 'other' forum) with some Pleurax a while back, but I cannot for the life of me recall who !

Mike

(*) as one of the three folk who started 'AD' I would say that !

[MicroBob]

Just this february we had a group meeting on diatom strew slide making and it was very well attended (19, I can only remember two meetings with more people attending) and the members and guests were very keen on trying their hand at strew slide making. We even overran the set time to work on one more sample. I wasn't sure whether this topic would really be a hit and was very happy to see that other people enjoy diatoms.
In times of climate crisis and the strong impact of micro algae on the world's photosythesis diatoms may move into focus more now. I generally see a tendency that people want to inform themselves practically and also to contribute to the research of biodiversity as citizen scientists. Our members of our group have set up a new project in cooperation with the university and a nature organisation to monitor the desmids in Hamburgs moors. This has started very well with a lot of participants. Unfortunately wherever you look: The species that were found a couple of decades before are to a large extent gone, not only with desmids.
It also appears that there are too few people today who are able to identify micro life under the microscope. This can make it difficult to compare old research results to new ones.

Bob

[frank/mic]

Hi
Is this combination a good one for plant section staining? If so could someone post a step by step method for this procedure please

Thanks

Frank