More on mild cleaning and incineration of diatoms

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Hobbyst46
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Re: More on mild cleaning and incineration of diatoms

#61 Post by Hobbyst46 » Wed Aug 15, 2018 6:59 pm

Here are some more photos of the diatoms, 36 hours after digestion with ~30% H2O2 in a boiling water bath. Most of the diatoms are clean except for a globule of pigment (lipid?). To achieve absolute cleanliness, I will add HCl, but I think that the point, that hot digestion with H2O2 is much more effective than cold digestion, has been demonstrated.

The specimens are the digested mixture, rinsed with DW.
Optics, from top to bottom: BF, 10X0.30 Neofluar; BF, 25X0.45 PLAN; POL, 40X0.75 Neofluar (the iridescent spots are mica/quartz debris); Phase contrast with blue filter, 40X0.75 Ph2 Neofluar.
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Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: More on mild cleaning and incineration of diatoms

#62 Post by Hobbyst46 » Wed Aug 22, 2018 4:26 pm

Here is a small "trick" I just "discovered" and I would like to share. Perhaps not everyone has seen it.
Alcohol (ethanol) is a known liquid medium for preservation of cleaned diatoms in suspension. It was also suggested as cleaning agent in a diatom cleaning protocol, but I can't remember where.
Some of my recent diatom batches, after mild cleanings, were somewhat unclean - in most of the frustules there were circular objects that looked like oil drops, bright green or golden. So, I replaced the water (after the last rinse) with ethanol (95%), and very shortly most of the frustules became glass-clear. Another weapon into the arsenal, cheap and ready.
P.S. I do not suggest to HEAT 95% ethanol. Ethanol vapors are irritating (at least).

MicroBob
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Location: Northern Germany

Re: More on mild cleaning and incineration of diatoms

#63 Post by MicroBob » Fri Aug 24, 2018 7:27 pm

Hi Doron,
this result looks really clean now! Not quite a mid cleaning anymore, but the result is good.
I too had the experience that ethanol takes on a yellow tint when diatom material is transferred to it. This tint is always a good sign that something is removed from the sample.

Keep you interesting work going and let us take part - always very interesting and well documented!

Bob

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: More on mild cleaning and incineration of diatoms

#64 Post by Hobbyst46 » Fri Aug 24, 2018 8:20 pm

MicroBob wrote:Hi Doron,
this result looks really clean now! Not quite a mid cleaning anymore, but the result is good.
I too had the experience that ethanol takes on a yellow tint when diatom material is transferred to it. This tint is always a good sign that something is removed from the sample.

Keep you interesting work going and let us take part - always very interesting and well documented!

Bob
Thanks Bob!
I have repeated the mild cleaning protocol (without acids) several times, although on only two quite similar samples of epiphytic diatoms.
The current protocol is:
a) EDTA+SDS (twice). Rinse well afterwards.
b) H2O2 in boiling water bath. 30% H2O2 is better, in that it removes much of the organic non-diatoms detritus. 3% H2O2 is easily available and cheap, but is less effective against the non-diatom detritus, like multicellular algae remnants, desmids etc. H2O2 can be applied twice.
HCl can be added at this point, but is not effective when cold, and I avoid heating it altogether.
c) Rinse and transfer to pure (95%) ethanol.

I intend to try to eliminate the residual organic detritus by gentle incineration - when preparing for mount, I will let the diatom suspension dry out on coverslips, than heat the coverslips on the metal plate (an idea I adopted from your protokol) very carefully, so the temperature remains below about 250C.

My future challenge in this field is diatoms in marine sediment sandstone rocks - if I find them! perhaps they will surrender to citric acid (hydrochloric is most probably better in all respects, except safety). And freeze-thaw cycles.

MicroBob
Posts: 3154
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: More on mild cleaning and incineration of diatoms

#65 Post by MicroBob » Sat Aug 25, 2018 8:34 am

Hi Doron,
in german mikroskopie-forum.de somebody showed a successful method for quick freeze-thaw cycles by using a Peltier element. The warm side of the Peltier has to be cooled very well, a CPU cooler works well. The freezing juice could bust brittle containers, so a metal foil container would be an idea. Peltier elements usually work with low voltage and high currents.

In Germany you can get H2O2 only up to 12% concentration as a private person. I plan to get sds and H2O2 in the future and try your recipe. All in all it is a good compromise between effort, aggressiveness of the substances and result.

Bob

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: More on mild cleaning and incineration of diatoms

#66 Post by Hobbyst46 » Sun Aug 26, 2018 12:56 pm

Bob,

Regretfully, I failed to read about the Peltier device in mikroskopie-de, since a guest is apparently not allowed to search that forum. Perhaps there is a way around it, a bypass that allows "direct" link to it?

12% H2O2 is definitely better than 3%, and if it is available in reasonable-size packages, so much the better.
Adding H2O2 to a crude diatom mass dilutes the H2O2 anyway. So, if possible, one should reduce the volume of the crude mass as much as practicable, e.g. remove the liquid phase by filtration through paper or dense mesh, than back-wash the retentate into a small volume of liquid. Or apply centrifugation (although this option is controversial).

I hope you had a successful diatom hunt in the countryside?

MicroBob
Posts: 3154
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: More on mild cleaning and incineration of diatoms

#67 Post by MicroBob » Sun Aug 26, 2018 8:34 pm

Hi Doron,
it was on www.mikroskopie-forum.de. Here is a link:https://www.mikroskopie-forum.de/index. ... ic=29862.0 I didn't find the link when I first looked for it. Can you see the thread?

I actually was not successful with diatom hunting, but I brought some sand samples with glittering particles in them.

Bob

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: More on mild cleaning and incineration of diatoms

#68 Post by Hobbyst46 » Sun Aug 26, 2018 10:00 pm

MicroBob wrote:Hi Doron,
it was on http://www.mikroskopie-forum.de. Here is a link:https://www.mikroskopie-forum.de/index. ... ic=29862.0 I didn't find the link when I first looked for it. Can you see the thread?

I actually was not successful with diatom hunting, but I brought some sand samples with glittering particles in them.

Bob
Thanks! I can see the thread and the links inside. Will translate and read.

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