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Cleaning diatoms
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Smokedaddy



Joined: 07 Oct 2006
Posts: 1680
Location: Phoenix, Arizona

PostPosted: Fri May 05, 2017 12:18 pm    Post subject: Cleaning diatoms Reply with quote

I was wondering if anyone has actually tried cleaning Diatoms with Liquid Drano that has Sodium Hypochlorite in it? I'm also interested in any other methods that are doable.

-JW:
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zzffnn



Joined: 22 May 2014
Posts: 1826
Location: Boston, Massachusetts, USA

PostPosted: Fri May 05, 2017 1:25 pm    Post subject: Reply with quote

JW,

We did try bleach and did not like the result (we saw incomplete cleaning and some breakage). The highly basic sodium hydroxide component of bleach can damage diatom, if soaking goes over 4 hrs.

The best way is to boil in highly concentrated (and highly dangerous) acids, such as 98% sulfuric acid. But I don't recommend this, unless you have handled such chemistry before.

We do not want to boil in acids, so we use the following procedure:

1) take diatom precipitates, add 31% HomeDepot muriatic acid and soak for 24-48 hrs;

2) add a few (start very slowly with one) very small drops of 30% hydrogen peroxide (3% won't work as well, you need 30%):
https://www.amazon.com/gp/aw/d/B01D3NTS12/ref=mp_s_a_1_5_a_it?ie=UTF8&qid=1487248928&sr=8-5&keywords=30+hydrogen+peroxide&dpPl=1&dpID=51RxCQM1pfL&ref=plSrch

3) then put in a few tiny HomeDepot potassium permanganate solids and soak for 24hrs (some use more toxic sodium dichromate in this step, with likely better result) ;

4) carefully wash/pour off acid, take precipitate, then add a few tiny drops of 35% hydrogen peroxide;

5) soak in muriatic acid again for 1 hr to remove any potassium permanganate or degradation product thereof;

6) wash with deionized water 4 times to remove all chemicals;

7) dry on cover slip, mount in Pleurax

Cool careful heating / invert drying for the ending half time ;

9) ringing with black enamel and label
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Last edited by zzffnn on Fri May 05, 2017 7:37 pm; edited 1 time in total
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Smokedaddy



Joined: 07 Oct 2006
Posts: 1680
Location: Phoenix, Arizona

PostPosted: Fri May 05, 2017 1:44 pm    Post subject: Reply with quote

Thanks, have you tried this method?

https://www.youtube.com/watch?v=CBEA1DF9BaA

-JW:
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zzffnn



Joined: 22 May 2014
Posts: 1826
Location: Boston, Massachusetts, USA

PostPosted: Fri May 05, 2017 2:05 pm    Post subject: Reply with quote

Smokedaddy wrote:
Thanks, have you tried this method?

https://www.youtube.com/watch?v=CBEA1DF9BaA

-JW:


That was highly similar to the bleach method that I tried. I actually read a research methodology paper describing the bleach method and follow it exactly. It does work, but results is noticeably worse than acid / oxidant soak.

Almost all the pros in the diatom field use acid boil. They won't do it, if bleach works as well.

Cold acid + oxidant soak is a much safer method than acid boil, and it still works reasonably well.

Try a few and pick one that works best for yourself :-p each sample/cleaner/operator combo is different.
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Last edited by zzffnn on Fri May 05, 2017 2:44 pm; edited 1 time in total
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zzffnn



Joined: 22 May 2014
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Location: Boston, Massachusetts, USA

PostPosted: Fri May 05, 2017 2:36 pm    Post subject: Reply with quote

Sorry, I forgot an important very first step:

Use a filter to remove everything larger than 0.4 mm (or 400 microns). Those are mostly minerals/sand particles and plant fibers.

For fossil diatom samples, you firstly need to repeatly freeze thaw them for at least 6 times. Then boil in strong acid. Cold acid soak won't work very well with fossil diatoms.
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Smokedaddy



Joined: 07 Oct 2006
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Location: Phoenix, Arizona

PostPosted: Fri May 05, 2017 2:49 pm    Post subject: Reply with quote

Thanks for the suggestions. As a novice and having done absolutely nothing like that before it seems rather dangerous but I appreciate the posting.

-JW:
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ChrisR
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Joined: 14 Mar 2009
Posts: 8518
Location: Near London, UK

PostPosted: Fri May 05, 2017 3:51 pm    Post subject: Reply with quote

Quote:
you firstly need to repeatedly freeze thaw them for at least 6 times.

What does that do? Crack them up?

The Home depot "muriatic"acid is approx 30% HCl in water. I've been pretty careless with that. I'm not recommending anyone copy, but I got it on my hands from time to time. At room temperature it washed off without effect, when warm it just made skin go pink for a short while.
(Don't store near anything metal though - very weak fumes will case rusting.)

I definitely wouldn't let that happen with sulphuric acid, nitric acid,.. Shocked Shocked
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zzffnn



Joined: 22 May 2014
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PostPosted: Fri May 05, 2017 4:08 pm    Post subject: Reply with quote

Yes and yes, ChrisR.

Freeze - thaw cycles break down the fossil rocks. Some do it for 10 times.

I selected muriatic acid for safety reason. It won't hurt you much for a short time, even on naked skin. You want to use gloves and avoid breathing in its vapor though, or at least breath in some tea vapor (tea is a base - the opposite of acid) to neutralize the acid vapor after handling it.

Low concentration sulfuric acid or nitric acid won't hurt that much either. But I have not tried on my skin what the upper limit is. If left on long enough though, they may dissolve meat/skin.

Basically:

I) acid works like animal stomach juice to digest organic tissues and reduce calcium/minerals;

II)Oxidants (bleach, hydrogen peroxide, potassium permanganate or sodium dichromate) help further breaking down organic tissues

III) steps 4-6 above remove all chemicals step by step.

Depending on how clean the original raw sample is, you may use less or more steps.

Some beach sand has naturally clean diatoms and need minimal cleaning, other than removing sand particles, for example.

Some freshwater river samples have lots of green organic stuff and may need a water boil (to white ash) after filtration.

Some fragile diatoms cannot survive water boiling or acid/oxidant soak though (they would disappear).
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Last edited by zzffnn on Fri May 05, 2017 7:36 pm; edited 1 time in total
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Charles Krebs



Joined: 01 Aug 2006
Posts: 5843
Location: Issaquah, WA USA

PostPosted: Fri May 05, 2017 5:24 pm    Post subject: Reply with quote

If you are really, really, serious about diatoms the cleaning methods used by the "pros" are pretty well documented. Personally, as someone more interested in "live" diatoms they are more involved than I care to pursue.

I suspect that zzffnn's reference to a bleach method "paper" may be this one.
https://tinyurl.com/zc36t3g

Some time ago I followed this procedure and found it too hard on the delicate part of the frustules. More recently I tried bleach again but this time carefully monitored the progress through a stereo microscope. The time it took to get very clear fustules was only a fraction of the recommended time in the "paper". The diatoms I tried this with were relatively large marine specimens, so it may not work well with small very delicate diatoms. I don't know.

My point is certainly not to disparage the tried and true methods used by serious diatomists. It is to encourage anyone who simply will not ever try the more complex (and somewhat more hazardous) methods to go ahead and try a basic bleach procedure. Just keep an eye on the progress and don't overdo it and you may be very happy with the results.

These diatom images I posted relatively recently were all from the last batch I cleaned using only bleach with water washes:

http://www.photomacrography.net/forum/viewtopic.php?t=33485
http://www.photomacrography.net/forum/viewtopic.php?t=33339
http://www.photomacrography.net/forum/viewtopic.php?t=33303
http://www.photomacrography.net/forum/viewtopic.php?t=33497
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zzffnn



Joined: 22 May 2014
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PostPosted: Fri May 05, 2017 5:51 pm    Post subject: Reply with quote

Yes, Charles. Observing bleach cleaning process can definitely work very well.

I just think the cold acid + oxidant method may be more consistent and less harsh, if the microscopist is comfortable with (and care to buy/use) those chemicals.

And that was the exact paper I followed. They cleaned some medium to big freshwater diatoms. Following their protocol, I observed bleached freshwater small diatoms at 2hr and 4hr after bleach soak, under a compound scope. Neither were cleaned enough for my sample and the 4hr sample had some delicate structures destroyed (cannot remember about how much was destroyed by 2hr treatment).

I think some delicate and complex structures in freshwater diatoms can be hard to cleaned completely and easy to destroy, at the same time.

Yes, some diatoms are very Hardy, while some are very fragile. I had some small marine one that disappeared after a hot water boil (not burn, just boil). But most seem to survive a burn just fine. Also some can be damaged by centrifugation

And it may depend on where sample comes from too. Coming fresh from slimy rock scrapping, such diatoms may have more organic tissues, which make them more difficult to clean. I had some Galveston marine rock scrapping that falls into this category.

Coming from within big sand particles (deeper deposit zones), some dead diatoms may already be in skeleton forms and need almost no cleaning. I had some Pensacola Florida sand diatoms that almost need minimal cleaning, as an example.

And the following is two examples of what the result looks like, using a similar procedure with sodium dichromate + hydrogen peroxide without acid or potassium permanganate (acid will produce even cleaner images).

This one came from brackish water, scrapping from submerged tree branch (in Armand Bayou, Clear Lake City, Texas)
http://www.microbehunter.com/microscopy-forum/viewtopic.php?f=6&t=3919&p=35756&hilit=bayou+Darkfield#p35756

This one came from beach sand of Pensacola Florida:
http://www.microbehunter.com/microscopy-forum/viewtopic.php?f=6&t=3962&p=36221&hilit=Pensacola+darkfield#p36221
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ChrisR
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PostPosted: Fri May 05, 2017 7:06 pm    Post subject: Reply with quote

just a quibble really - don't confuse sulfonic acid (or sulphamic acid) with sulfuric/sulphuric acid/H2SO4. The last of those is readily available, but a jump up in care is needed.
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zzffnn



Joined: 22 May 2014
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PostPosted: Fri May 05, 2017 7:40 pm    Post subject: Reply with quote

ChrisR wrote:
just a quibble really - don't confuse sulfonic acid (or sulphamic acid) with sulfuric/sulphuric acid. The last of those is readily available, but a jump up in care is needed.


Thanks ChrisR and nice save/catch! I meant sulfuric acid (with H hydrogen) but typed down sulfonic acid (which has R - alkyl or aryl - instead of H) Shocked - sorry for my brain fart or finger spasm Embarassed Sulfonic acid won't be acidic enough to clean diatoms in two days.

I have corrected those two typos.
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Beatsy



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PostPosted: Sat May 06, 2017 1:46 am    Post subject: Reply with quote

I always use the boiling H2SO4 method for fossil material (diatomite), it's by far the most predictable and reliable in my experience. With an initial soak in HCl to remove carbonates and dissolved metals before the acid boil. Use small quantities, treat it with respect and do most of it outdoors and it's not that hazardous. For "squeaky clean" frustules a shock 30-second boil in dilute (0.5% w/v) NaOH, then quickly neutralise with HCl displaces the last bits of detritus. It can dissolve delicate forms though so adjust time accordingly. And boiling caustic is more dangerous than boiling acid! So be extra careful with that one.

For fresh samples, sub-fossil material and some mudstones you can do away with the acids and alkali altogether. Just a very long boil (2-6 hours) in DI water, H202 with a pinch of TSPP (tetrasodium pyrophosphate) will often do a great job. I use a glass milk-pan with a plate over the top rather than a beaker or flask as it handles the inevitable "bumping" better. Mudstone samples may need an NaOH shock at the end, but usually not.

Note: I wouldn't advise extracting larger (0.4mm) lumps earlier in the process. These are *very* often clumps of diatom frustules that are loosely welded together by dissolved silica. The NaOH shock breaks those up.
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75RR



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PostPosted: Sat May 06, 2017 3:34 am    Post subject: Reply with quote

Here is a long and yet very interesting thread that you might want to mine for information on this:

http://www.microbehunter.com/microscopy-forum/viewtopic.php?f=10&t=3036
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zzffnn



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PostPosted: Sat May 06, 2017 5:59 am    Post subject: Reply with quote

Beatsy wrote:
........For fresh samples, sub-fossil material and some mudstones you can do away with the acids and alkali altogether. Just a very long boil (2-6 hours) in DI water, H202 with a pinch of TSPP (tetrasodium pyrophosphate) will often do a great job. I use a glass milk-pan with a plate over the top rather than a beaker or flask as it handles the inevitable "bumping" better. Mudstone samples may need an NaOH shock at the end, but usually not.

Note: I wouldn't advise extracting larger (0.4mm) lumps earlier in the process. These are *very* often clumps of diatom frustules that are loosely welded together by dissolved silica. The NaOH shock breaks those up.


Beatsy,

Do you think a pressure cooker cook can reduce the boiling time? 30 min in my pressure cooker is about the same as 2 hrs of boiling.

The thing with boiling >2hr is that, I either would forget about it (fire hazard!) or cannot wait that long. Spill over may be a concern with pressure cooker though.

Re your recommendation against filtering with 400 micron pores, does that apply to fossil only or fresh sample as well?

I guess we can increase pore size to say 1mm and/or filter after chemical treatment. I have a slight worry that too much organics lumps may reduce the chemical's cleaning power though. But 6hr water boil should take care of it.
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