An experimental convenient alternative method for cleaning epiphytic diatoms

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Hobbyst46
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An experimental convenient alternative method for cleaning epiphytic diatoms

#1 Post by Hobbyst46 » Thu Sep 26, 2019 4:16 pm

Ammonium persulfate (APS) is a white powder, free-flowing, easy to handle, stable at ambient temperature, relatively safe, non-odorous, very soluble in water, and very inexpensive. AFAIK, it has not been considered for diatom cleaning; at least, it does not star in the popular protocols. Here are the results of a first experiment: I cleaned a raw live diatom suspension from decaying freshwater plants and algae by boiling with APS in water for 2-3 hours. Roughly 1/3-1/2 of the diatoms were totally cleaned by this treatment, while others still contained tiny amounts of organic matter (golden-brown globules). A second iteration lead to nearly-all clean frustules.

Below are single phase contrast images, taken with 100X1.3 Planapo (raw sample) and 40X0.75 Neofluar (cleaned). Samples are wet mounts in water. The prominent diatom is Rhopalodia gibba (just my guess). It is 80-90um long.
APS cleaning appears to be gentle, even if slightly less than complete. Frustules remained intact (this is an advantage/disadvantage, depending on the microscopist...) ;)
This is not as yet a suggested protocol, because: (1) the efficacy is demonstrated here only with one pond slime sample, (2) the optimal amount of APS per batch is not yet established. Too much APS might interfere with precipitation and settling of the frustules after the treatment.

Comments are welcome.
Attachments
Raw sample.JPG
Raw sample.JPG (55.17 KiB) Viewed 1053 times
Post 1st APS treatment (1).JPG
Post 1st APS treatment (1).JPG (40.34 KiB) Viewed 1053 times
Post 1st APS treatment (2).JPG
Post 1st APS treatment (2).JPG (32.88 KiB) Viewed 1053 times
Post 1st APS treatment (3).JPG
Post 1st APS treatment (3).JPG (45.09 KiB) Viewed 1053 times
Post 2nd APS treatment.JPG
Post 2nd APS treatment.JPG (40.48 KiB) Viewed 1053 times
Last edited by Hobbyst46 on Thu Sep 26, 2019 5:49 pm, edited 1 time in total.
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mnmyco
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#2 Post by mnmyco » Thu Sep 26, 2019 5:14 pm

A great preliminary result.

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#3 Post by Hobbyst46 » Thu Sep 26, 2019 5:50 pm

Thanks mnmyco.
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MicroBob
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#4 Post by MicroBob » Thu Sep 26, 2019 6:42 pm

Hi Doron,
great to hear you are in the laboratory again! The results look really good.
ammonium persulphate is a bit difficult to get in Germany which is probably a sign that it is good stuff.
Wereabouts is the pH value of the dissolved powder?
It has been in use in circuit board etching.

Your golden-brown globules could be oil drops so they might be easy to remove with an intermediate step in e.g. lighter fluid.

This can really be an interesting method - I don't really care for the last % of performance as long as it is suitable for the home lab.

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#5 Post by Hobbyst46 » Thu Sep 26, 2019 7:51 pm

MicroBob wrote:
Thu Sep 26, 2019 6:42 pm
Hi Doron,
great to hear you are in the laboratory again! The results look really good.
ammonium persulphate is a bit difficult to get in Germany which is probably a sign that it is good stuff.
Wereabouts is the pH value of the dissolved powder?
It has been in use in circuit board etching.

Your golden-brown globules could be oil drops so they might be easy to remove with an intermediate step in e.g. lighter fluid.

This can really be an interesting method - I don't really care for the last % of performance as long as it is suitable for the home lab.

Bob
Thanks Bob,
Actually, I returned to specimens only after I fixed the X and Y movement of the old stage (thanks for directions).
The pH of the dissolved powder is 1-2 according to literature and I verified that this is so. In my diatom treatment liquer the pH was probably 2-3.
APS works over a broad pH range, 3-7 at least. A pH of 3 should be fine. No need to add acid or base, since the raw diatom suspension in DW does not contribute to pH. I do not know if HCL supports the action of APS or not - probably not.
Cold, pure APS solution reacts too slowly to be effective. It needs either heat, or the presence of metal ions, e.g. silver or iron for its action.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#6 Post by MicroBob » Thu Sep 26, 2019 8:21 pm

So it is not in the basic region which could be a problem - much better than Clorix then.
For ciruit boards it is used at about 50° for 1-2 minutes, so it attacks the copper very aggressively.
I read that the solution should not be stored in a completely closed bottle as it emits oxygen.
I'm very interested in your further results, this can really be an especially attractive method for the home lab amateur microscopist.

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#7 Post by Hobbyst46 » Fri Sep 27, 2019 6:53 am

MicroBob wrote:
Thu Sep 26, 2019 8:21 pm
So it is not in the basic region which could be a problem - much better than Clorix then.
For ciruit boards it is used at about 50° for 1-2 minutes, so it attacks the copper very aggressively.
I read that the solution should not be stored in a completely closed bottle as it emits oxygen.
I'm very interested in your further results, this can really be an especially attractive method for the home lab amateur microscopist.

Bob
APS solution corrodes copper and iron. Glass is OK. Falkon tube is OK.
I store the solid APS powder at room temperature in a tightly closed jar (to keep it dry, prevent humidity). Make as much solution as needed in a loosely caped flask or bottle. Use the solution right away. Again, this is not a protocol. Please note, however, that LIKE IN SIMILAR EXPERIMENTS, eye protection (closed goggles) and gloves (thin rubber is fine) are a must. APS powder and solution are a serious eye irritant, skin irritant and respiratory tract irritant (although better than 30% H2O2, AFAIK). For 1-2ml of raw diatoms, about a teaspoonful of APS does the job.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#8 Post by MicroBob » Sat Oct 05, 2019 1:40 pm

Hi Doron,
I have just received my APS from Poland. In July we were on holiday here: 53°11'59.5"N 12°54'17.9"E
I collected there a big diatom rich sample by scaping of water plants. This is probably just the right material for a test.

To dissolve Orcein in 45% acetic acid I just used the shown setup. The little hot plate reaches 120°C, not enough to boil the acetic acid-water mix but hot enough to dissolve the orcein.
My "laboratory equipment" is very limited, there is always something I would need but don't have. :roll:
My "laboratory" is my new outside workshop with transparent roof. Too cold in winter but very nice for the rest of the year and directly in front of the workshop.
This setup will probably do fine for applying hot APS - I will report.

Bob
Attachments
Auflösen Orcein-Essigsäure 1024.jpg
Auflösen Orcein-Essigsäure 1024.jpg (174.78 KiB) Viewed 935 times

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#9 Post by Hobbyst46 » Sat Oct 05, 2019 2:29 pm

Hi Bob,
Congratulations on the successful home rennovation and founding of a home lab.
Having only the minimum must-have items keeps the bench uncluttered.
A very nice view from the lab as I can see!

Note that those red rubber tube connectors are not resistant against all organic solvents. Room-temp Alcohols, petrol ether, acetone are OK; paint thinner, toluene and xylene - not so good, inspect the rubber periodically for drying and cracks.

As for the APS - still, I did not write a protocol. I have repeated the treatment once more on another portion of the same collected raw freshwater sample, and it worked OK. I am amazed that you take the chance to try it!

Note, though, that APS is not very efficient against non-diatom debris - even an iteration of the process does not destroy all of it.

For APS, use goggles and gloves (thin nitrile rubber are fine). Note the concentration. A very high concentration is not good IMO.
I suggest about 6-10ml of a 50-60% solution of APS in water. Add it portion-wise to 25ml of the live diatom slurry in a 100ml conical flask. The pH of this slurry is 1-2. The bend glass tube that leads to a splash/vapor trap is OK if not blocked by condensed liquid.

Heat the mixture on the hotplate, so it should reach the boiling point within 30-45 minutes. The color should change from the murky green/brown to pinkish-yellowish-of white. But it can take 3+ hours. Add water to prevent the slurry from drying. Note that both APS and its decomposition products are very soluble in water. Afterwards, rinse many times.

P.S. add a boiling grain ("boiling stone") to the diatom in APS slurry, if you have one (as a bubbling source). Or, if your hotplate is a stirrer as well, add a teflon-coated magnetic stirring bar.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#10 Post by MicroBob » Sat Oct 05, 2019 5:05 pm

Hi Doron,
I had the feeling that APS might be a very promising candidate so I wanted to try myself. For making strew slides of a certain sampling location I don't find it necessary to get rid of every little piece of dirt as along as the diatom frustules are clean and clearly visible.
To get a sample completely clean always more than one method has to be applied, at least to remove sand particles. What would you suggest as supportive cleaning steps for APS to clean the sample I have at hand? It is from fresh water, mostly scraped from the long stems of water lilys in a muddy bay. So no salt, nearly no sand, diatoms with oil drops, some rotting plant tissue.

- bathing in acetone in a 25µ sieve? Before or after APS treatment?
- mixing with water after APS and give frustues just enough time to settle before the dirt settles too?

Thank you for the hint with the boiling stone - I probably will take a small stone from the garden.
My heat plate is just an aluminium block and the simple regulator doesn't allow more than 120°C.
With the APS I bought a heat transmitting pad, I hope this makes heating fluids on the little heat plate easier.

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#11 Post by Hobbyst46 » Sat Oct 05, 2019 6:27 pm

MicroBob wrote:What would you suggest as supportive cleaning steps for APS to clean the sample I have at hand? It is from fresh water, mostly scraped from the long stems of water lilys in a muddy bay. So no salt, nearly no sand, diatoms with oil drops, some rotting plant tissue.
- bathing in acetone in a 25µ sieve? Before or after APS treatment?
- mixing with water after APS and give frustues just enough time to settle before the dirt settles too?
Neither. Maybe just wet sieving through a 300-500um mesh (assuming that the diatoms are not that large) to remove plant tissue. I myself am mentally working on a supportive step, for this very type of sample. Already tried 3% hypochlorite in the cold, as well as hot hypochlorite, but neither of them decomposes cellulose, so plant cell walls remain intact, and I am still thinking about it...
Thank you for the hint with the boiling stone - I probably will take a small stone from the garden.
Not a garden stone !!! if it is a carbonate, it will ruin your experiment!!! since the pH is acidic !!!it should be a porous material. A small piece of unglazed porcelain might do.
My heat plate is just an aluminium block and the simple regulator doesn't allow more than 120°C.
With the APS I bought a heat transmitting pad, I hope this makes heating fluids on the little heat plate easier.
Perhaps, instead of the aluminum block, you can immerse the flask in a boiling water bath.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#12 Post by MicroBob » Sat Oct 05, 2019 7:16 pm

Hobbyst46 wrote:
Sat Oct 05, 2019 6:27 pm
Not a garden stone !!! if it is a carbonate, it will ruin your experiment!!! since the pH is acidic !!![/u][/b][/color]it should be a porous material. A small piece of unglazed porcelain might do.
The stones in your and my garden are probably quite different. Calerous stones would break up here due to the frost and become part of the soil. What I have in mind is a pebble like a bigger sand grain - these should be acid resitant - I will try first. I have some porcelain rings used in high temperature electrical isolation, these should work too.
For heating fluids it would be nice to be able to place the temperature feeler directly in the water bath but if it would fall out the hot plate could reach dangerous temparature regions, so I leave it as it is. Maybe I will paint the block with heat resistant paint to increase heat radiation.

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#13 Post by Hobbyst46 » Sat Oct 05, 2019 7:53 pm

MicroBob wrote:
Sat Oct 05, 2019 7:16 pm
For heating fluids it would be nice to be able to place the temperature feeler directly in the water bath but if it would fall out the hot plate could reach dangerous temparature regions, so I leave it as it is.
Is this temperature feeler (sensor) a part of the heat controller of the aluminum block ? meaning that the PID (or a simpler controller) responds to this sensor ? in that case, then the sensor IMO should NOT be in the water. I would drill a small hole in the block surface and forcibly stick about 1-2cm of the sensor tip inside. Then secure the sensor somehow to prevent it from falling out. This way it will control the block and serve as a hotplate. Especially if it is a PID, the way to get reproducible and predictable control is that the sensor feels the temperature of the directly heated element, not something that is isolated from it (in this case, by the glass wall of the flask). 120C is rather low for a hotplate; models that I have known reached 250-300C (I think). So your APS solution might be warmed up to 50-60 maybe. Hopefully my guesses are pessimistic and it will work well....But heat losses from all around the flask walls are against it...

I have built some small heating aluminum blocks, using a "finger" heating resistance element of 6mm diameter in a stainless steel sleeve; if memory works, it was a 200-300W "finger" but the block was smaller than the one you show above. A decent lab hotplate boasts 800W at least.

P.S. I might also try a 1.5 liter ordinary metal pot full of boiling water and standing on a tripod over an alcohol burner. Immerse the conical flask in it (before the burner flame is on!) and stabilize it by means of a clamp. As an alternative, if the hot plate is found to be too weak (hopefully not!).
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#14 Post by MicroBob » Sat Oct 05, 2019 8:31 pm

I have two hot plates:
1. simple controller up to 120°C, 40W heat element from 3D printer head, temperature feeler gued in with heat transmitting glue, aluminium block (see picture), about 1,5x5x10cm.
I can't remeber the values but there was a lot of difference between hot plate temperature and sample temperature but is was not too bad. But this will be increasingly difficult with rising temperature leel.
2. the one I made a post about here, 800W, PID controller, up to (questionable for safety reasons) 350 °C or so.

If #1 and #2 won't do it I would have to take #3 or #4 :lol:
There are a little cooking plate (could be controlled with an external power controller, no thermostat).
And a travel clothes iron with foldable handle, will probably cook very well on "Linen" setting. :lol:

I read about APS in circuit board etching that it is useless without heating, but good at 50 °C. So it might work very well for diatom cleaning at a moderate temperature like 80°C.

According the mixture:
"I suggest about 6-10ml of a 50-60% solution of APS in water. Add it portion-wise to 25ml of the live diatom slurry"
This would be then about a tea spoon full onto 33ml of diatoms in water - sounds good to me. Is it useful to dissolve APS in water before adding to the diatom slurry?

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#15 Post by Hobbyst46 » Sat Oct 05, 2019 9:06 pm

About the hotplate - #2 IMO will do the job. #1 will not, IMHO.

It is covenient to dissolve the solid APS in a little water - say, ~5ml. APS can be made to ~60% in water at a temperature of 25C. When you mix it with water, the liquid becomes cooler, because this dissolution is endothermic. Dissolution is fast, though, and is complete within minutes. Then add the solution dropwise to the diatom slurry. That is how I did it.

But, I am sure that it is OK to just pour (slowly! use a powder funnel if you have one) the powder into the diatom slurry, and slowly mix it with a glass rod. Dissolution will be fast, although if the liquid is turbid, crystals are invisible anyway. Then, at room temperature, nothing seems to happen. Cap the flask with the cork-and-bent-tube assembly and heat. Oxygen is released, along with water vapor, so do not let overpressure develop.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#16 Post by Hobbyst46 » Mon Oct 07, 2019 1:46 pm

Forth and fifth cleaning experiments. These ones were performed on fresh slurries of marine diatoms. These were obtained from marine algae by mechanical manipulations into water as described above. The slurry was filtered through a 0.3-0.5mm mesh. The filtrate was filtered through coarse filter paper, to get read of as much of the seawater as possible (those salts may possibly interfere with the oxidation). This latter filtration was slow, ~30h. The residue on the paper was rinsed with distilled water into a flask, to form the slurry. To the flask I added solid APS as above and immersed it in a bath of boiling water (temperature within the flask - 95C) for 1.5-2h. Then placed the flask directly over a small alcohol burner and boiled it down to a volume of 15ml.
The two experiments yielded practically the same result, the photos below refer to one of them.
The first two photos show the samples of raw slurry and clean slurry, respectively (total volume of each in the vial).
Next, images of live diatoms, in distilled water, among a large amount of debris.
Then images of clean diatoms in distilled water. The relative amount of debris decreased, and many of the diatoms were clean. I was happy to find some new (to me!) species by the way.

One important note about APS: In the process, it decomposes into acid and sulphate. If the diatom slurry initially contains mineral calcium, e.g. calcium carbonate, this might lead to formation of calcium sulphate, which is poorly soluble in water, so might precipitate and ruin the whole sample. Epiphytic diatoms are relatively free of mineral. Hopefully the process can be used with benthic diatoms as well, after an initial SDS+EDTA treatment (so the APS will replace the weak H2O2 step).
Attachments
raw diatom suspension.jpg
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post-APS diatom suspension.jpg
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Last edited by Hobbyst46 on Thu Oct 24, 2019 7:25 pm, edited 1 time in total.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#17 Post by Hobbyst46 » Thu Oct 24, 2019 7:12 pm

(continued) - raw diatoms
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#18 Post by Hobbyst46 » Thu Oct 24, 2019 7:18 pm

(continued) - post cleaning.

Note: These results are actually after two cycles of heating with APS. I am not sure, however, that the second stage contributed significantly. Likewise, perhaps the heating in a water bath does the job, I am not sure that boiling is essential. But it is easy to perform, so for the time being, I do it.
The second image here shows a suspicious form. Is it a diatom ? cannot tell. Note that all photos are diatoms in water, not Pleurax. I saw other symmetrical forms, a sort of double-pointed bobbin, sizes <100um but cannot identify them.
Attachments
220um long (maybe Licmophora colosallis).JPG
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A diatom or an unknown.JPG
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Diploneis stack of 2.jpg
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long diatom and Grammatophora (1).JPG
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long diatom and Grammatophora (2).JPG
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#19 Post by MicroBob » Thu Oct 24, 2019 7:37 pm

Hi Doron,
very impressive results! The picture of the second bottle says it all: Clean! If there were remaining dirt it would not have this silvery white look.
As a first step I would try to get rid of salt (which you did too) and calcerous matter, which EDTA or HCL or concentrated vingar could do.
Couldn't this step be combined with the washing with destilled or demineralized water? So not wash with water in the filter but an acid ?
For a quick image a dry preparation could be useful: The difference of the r.i. of diatoms (1,4-1,5) and air (1,0) is big so there is a lot of contrast.
I was quite astonished that the dried out salt water slide here: viewtopic.php?f=6&t=8130 gave such a nice image.
The unidentified object could be a sponge needle, but I'm not sure.
My carpenty is done, now a bit plumbing and my outdoor lab is free for an APS test!

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#20 Post by Hobbyst46 » Thu Oct 24, 2019 8:35 pm

MicroBob wrote:
Thu Oct 24, 2019 7:37 pm
very impressive results! The picture of the second bottle says it all: Clean! If there were remaining dirt it would not have this silvery white look. As a first step I would try to get rid of salt (which you did too) and calcerous matter, which EDTA or HCL or concentrated vingar could do.
Couldn't this step be combined with the washing with destilled or demineralized water? So not wash with water in the filter but an acid ?
For a quick image a dry preparation could be useful: The difference of the r.i. of diatoms (1,4-1,5) and air (1,0) is big so there is a lot of contrast.
I was quite astonished that the dried out salt water slide here: viewtopic.php?f=6&t=8130 gave such a nice image.
The unidentified object could be a sponge needle, but I'm not sure.
Thanks Bob.

1. If the initial raw mixture is in distilled water (DW) or tap water (TP) it makes sense to add acid or EDTA to the mixture (on calcium carbonate, acid will work much better than EDTA, because calcium carbonate is insoluble), let it act on the minerals for some time, then filter. Or, alternatively, combine them as you suggest. The only drawback is the foul smell and potential harm of the acid vapors that emanate from itself the filtration funnel+paper for many hours. Both HCL and acetic acid vapors are harmful. Vacuum-assisted filtration can accelerate the process, but to many minutes, not seconds.

2. If the initial raw mixture is natural water, I would not apply acid (nor APS!!) directly, but rather transfer the diatoms to DW or TP then apply the active chemical. That is because natural water might contain stuff which releases unwieldy vapors.

3. In practically all methods of oxidation and decomposition of the detritus and organic diatom contents, it is important to remove the HCL or acetic acid or EDTA before the addition of APS, otherwise the APS will be inhibited or wasted by acting on the acid instead of the target detritus.

4. Agreed about mounting in air; the images above are just quick results, phase contrast images so I thought that water is still OK.

Side note: I find the software Toupview very friendly for simple stacking of JPG files.

P.S. some say that APS is not very stable, since if it (as powder) absorbs water from ambient humidity, it gradually decomposes, so loses its activity over a period of months (depending on the ambient conditions). As a preventive measure, I would store APS in hermetically-closed jars. Better yet, divide the purchased APS into small portions (say, if I purchased 50g, divide it into 10 5g portions) and store them in individual sealed containers until use.
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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#21 Post by MicroBob » Fri Oct 25, 2019 3:44 pm

Hi Doron,
thank you for clarification!
So I would use this process for samples in sea or fresh water:

1. remove unnecessary water and replace with DW by rinsing in a filter
2. Apply HCL outside to get rid of calcerous matter
3. Rinse again to remove HCL
4. Apply APS for 1-2 hours hot
5. Cook down sample to a nice concentration (though bubbling could lead to breakage of sensitive frustules)
6. Remove APS by rinsing with DW

Thank you for the hint with the storage of APS. My 1kg APS came in a PE bottle that will not prevent decay forever. So I will fill it into glass jars with metal screw lids and divide it up into a small jar for active use and a bigger storage jar.

Bob

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Re: An experimental convenient alternative method for cleaning epiphytic diatoms

#22 Post by Hobbyst46 » Sat Oct 26, 2019 9:06 pm

I have just re-found an article that I found and downloaded a year ago, "Description and comparison of a new cleaning method of diatom frustules for light and electron microscope
studies" by JOSTEN C. W. MA and LELA M. JEFFREY, Department of Oceanography, Texas A&M University, College Station, Texas 77843, U.S.A.
Published in Journal of Microscopy, Vol. 112, Pt 2, March 1978, pp. 235-238.


These authors used potassium persulphate (PPS) in a single step of diatom cleaning. They used less than 1ug of PPS per ml (!?) of raw diatoms in water, heated it to 60-90C, for 6-8 hours, and state that the results were fine. I use large amounts of APS, perhaps unnecessarily; APS is much better soluble than PPS in water. Another persulphate of potential interest is sodium persulphate (SPS), which is almost as soluble as APS. Some authors claim that for water decontamination, SPS is better than APS. Also, APS yields buffered acidic solution (pH~2), whereas SPS and PPS do not (how relevant are these differences to diatom cleaning I do not know).

Diatom cleaning seems to remain of interest. Example: "A rapid cleaning method for diatoms" by Rosa Trobajo & David G. Mann, in Diatom Research, 34:2, 115-124,2019.
This article presents a table of many known methods of cleaning.
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