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PostPosted: Tue Jul 10, 2018 12:10 am 
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The oven thermometer simply sat over cooking pan by gravity; there was no thermo-coupling in between, so some heat was lost there. Temperature shown in the photo was not the highest achieved one.

The oven thermometer looks similar to this one, though temperature rating may be different: https://sc01.alicdn.com/kf/HTB15ViCIXXX ... 50x350.jpg

My stove flame took about 12 minutes to heat the thermometer to 325C, at highest temperature setting. Temperature rose very quickly then slowed down significantly at 325C. I let it sat at 325C for about 3 minutes, did not see temperature rising higher, so turned off the flame. Not sure if it will get higher, if I wait longer.

As you can see, that carbonized cooking oil drip was still there at 300-325C. So it may be better to incinerate after chemical cleaning, to reduce that charcoals formation.

Do you think cast iron cooking pans would work? I don't know how much iron oxide will be produced by heating cast iron at 400C. Iron oxide can be dissolved by by soaking in strong acid overnight and then wash by distilled water, if necessary.


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PostPosted: Tue Jul 10, 2018 9:19 am 
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zzffnn wrote:
My stove flame took about 12 minutes to heat the thermometer to 325C, at highest temperature setting. Temperature rose very quickly then slowed down significantly at 325C. I let it sat at 325C for about 3 minutes, did not see temperature rising higher, so turned off the flame. Not sure if it will get higher, if I wait longer.
Temperature will rise until the rate of energy input from the flame to the pan will equal the rate of heat loss to the surrounding. So, it will not get much higher. And, if the sensor tip - the tip of the small stick attached to the dial indicator - does not touch the surface, the actual temperature of the pan was probably higher than indicated, possibly by 20-40C.

Quote:
So it may be better to incinerate after chemical cleaning, to reduce that charcoals formation.
While I believe this is true, I became curious to try the other option (that you had suggested) as well. "Just" need to collect some fresh live diatoms.

Quote:
Do you think cast iron cooking pans would work?
Yes. Although cookware are made of various metal alloys, engineering data show, that in general, cast iron conducts heat better than stainless steel, whereas their specific heats are quite close to each other. BUT, if the weight of the cast iron is much higher than that of a stainless steel pan (for comparison), the time to reach a stable temperature on the cast iron will be much longer.
Also, we are talking bare, uncoated cast iron, is that right?

Quote:
I don't know how much iron oxide will be produced by heating cast iron at 400C.
Sorry, neither do I. It may depend on ambient humidity as well. The point is, that iron oxide will form repeatedly, every time you heat the pan, since "rust" layers are feebly attached to the iron body and tend to be porous.
Quote:
Iron oxide can be dissolved by by soaking in strong acid overnight and then wash by distilled water, if necessary.
Yes, but a strong acid will dissolve the iron core as well, overnight... besides, iron oxides easily dissolve in dilute mineral acids and in some organic acids, like citric acid (on the spices shelf).
So, I would do as follows: Having scraped the incinerated residue off the pan, I would try to clean the pan with citric acid or even vinegar. No point in trying to remove every rust molecule - it will reappear in the next incineration.

And most importantly: to remove iron oxides from the diatoms, use either dilute HCl (say 5-10%), or citric acid, it will be a quick process - minutes I believe. Dilute H2SO4 will do the job of course as well.
If you decide to use a strong acid on the pan itself (which I do not recommend) please leave it for minutes, not hours.
Another funny option to remove rust from diatoms is to dip a clean small magnet in the aqueous suspension of the diatoms, since iron oxides are magnetic...

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PostPosted: Tue Jul 10, 2018 12:01 pm 
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Tip of my oven thermometer touched my cooking pan and rested on it by gravity. But I guessing there may be tiny air gaps at the touching surface and some heat might be lost going up from the tip to dial?

Sorry, I was not clear; I did not mean removing iron oxide from (uncoated) cast iron pan. I only meant removing them from diatom sample.


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PostPosted: Tue Jul 10, 2018 12:57 pm 
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zzffnn wrote:
Tip of my oven thermometer touched my cooking pan and rested on it by gravity. But I guessing there may be tiny air gaps at the touching surface and some heat might be lost going up from the tip to dial?
Yes, the thermometer measures accurately when the tip is inserted in an oven, not touching the inner walls, but the air inside is a constant temperature bath; also, the thermometer measures accurately when the tip is inserted into liquid in a cup, or into a closely fitting bore in a solid metal block. These are not the cases when the tip is barely touching a surface, because temperature gradient is large. Hence, I agree that the actual pan surface temperature was higher than what your thermometer indicated. And from experience I guess, that it could be higher by 20-40C.
It is possible to measure the pan temperature more accurately if you can find or make a small ALUMINUM block, say a cube or cylinder, size roughly 1"x1"x1". Drill a 1/2" deep hole to accept the thermometer tip as closely as practicable. Place the block flat on the pan to ensure good contact.
Copper is even better than aluminum, but drilling in copper can be tricky. Brass is an option. Silver and gold would be excellent... 8-)

P.S. Discovered a relatively cheap eBay source for zinc pellets to serve as temperature indicators (melting point: 419C):
https://www.ebay.com/itm/5-grams-Zinc-m ... SwCQZZCbcA

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PostPosted: Sat Jul 14, 2018 2:10 pm 
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Following are some experiments of incineration prior to cleaning with chemicals.

I collected some more epiphytic pond diatoms, including what I thought were submerged decaying plants, but introduced a much dark brown dirt and mud - that is, clay, silt etc. Thus the sample was actually quite poor in both diatom variety and total quantity. Nevertheless, I processed it. Wrung into water, filtered the mass through a 600 micron sieve and attempted filtration of the filtrate through a 25 micron filter cloth. The latter step was inefficient, since a "mud cake" formed on the filter and blocked it. I reverse rinsed the cake into water, mixed and waited for separation. laid drops of the dark brown sediment suspension were laid on top of 24x60 mm coverslips, 5-6 drops per coverslip. The coverslips were laid to dry in petri dishes, the lids being supported on stand-offs (photo 1). I incinerated them for 50 minutes on the gas stove as shown (photo 2). The temperature was less than 400C. The residue was a pitch-black coating that adheres to the glass but can be easily scraped in the form of powder.

I treated a sample of the powder with excess EDTA+SDS, and interestingly, the appearance of the liquid phase changed from colorless to pale yellow (photo 3). The solid phase turned dark gray. The yellow coloration indicates that some cleaning of either or both diatoms and non-diatom debris occurred. Next, I treated it with 3% H2O2 in a boiling water bath, but apparently without further changes. I rinsed it and prepared a wet mount with DW.

Photos 4 to 14 show the results. They are not as sharp and clear as I would have liked. The BF 10X are included to demonstrate the proportion of diatoms in the mass. They are mostly needle like (Synedra, {not Ulnaria} ? ) with others (Gomponema?), mostly smaller than 40 microns. The pressure of the 100X oil objective (with oil of course) on the coverslip tended to push the diatoms and move them in the FOV. I refer to non-diatoms here as "silt".

I also inspected an original incinerated-only sample, which had been heated for 25 minutes. I inverted the original coverslip on a slide, to make an air mount. Also, dispersed the black powder in powder and made a DW mount. The results (photos 15-19) show less bent or otherwise deformed diatoms, but many partially-clean diatoms.

Of course, the incineration done here is less controlled than in an oven, and there are temperature gradients on the heating plate. Also, the data are not
statistically based, and are limited to a few species. On the other hand, I strive to be objective, not pick out a specific diatom to prove a claim. So I very cautiously suggest the following conclusions:

1. Incineration softened and deformed diatoms. The Synedra are bent, other diatoms lost their perfect bilateral symmetry. So they were cleaned, but damaged. One might wonder, were the "glass" diatoms slowly melting at temperatures <400C ? I speculate that the presence of organic molecules as part of the diatom silica frame contributes to the mechanical stability of the frame. Chemical cleaning alone perhaps removes most or all of the organic components in a way that does not deform the frustules. Incineration perhaps destroys other components, that are critical to the stability of the frustules.

2. Incineration followed by cleaning cleaned many diatoms. Incineration alone, without chemical cleaning, left many unclean diatoms, see photos 15-19. I marked with red circles what I think is organic matter inside the frustule, not external. Even softened and deformed diatoms were not absolutely clean of organic contents - See, for example, photo 15.

3. Incineration promoted fracture, at least of the long Synedra. This hypothesis is based on my many other experiments so far with such pond diatoms.

4. The residue after the above described incineration is a powder that can be scraped, rather than tar lumps. So it should be possible to treat it chemically.

5. Incineration alone as a cleaning method is questionable: long term and high temperature heating softens and deforms the diatoms, short term and low temperature heating is less likely to damage, but apparently, less effective for cleaning. This opinion will probably meet some objection - I will take my chance... :)

6. I guess, that the post-incineration mass is black due to iron oxides plus charcoal. It was cleaned somewhat by the EDTA+SDS, presumably the iron was attached to EDTA.

Based on this experience, I would try to incinerate at a lower temperature, say around 300C, for less than 30 minutes, then clean it with chemicals. So the frying pan as shown by zzffnn might work do the job. The collected diatom sample described above is maybe a harsh test, containing too much non-diatom stuff, like mica, silt etc. Incineration of an initial plant-only sample might produce better results. I would give it a chance, and see what H2O2 will do to the incinerated mass.


Attachments:
(1) Dried - before incineration.jpg
(1) Dried - before incineration.jpg [ 132.44 KiB | Viewed 902 times ]
(2) 50minute Incineration - temperature below 400C.jpg
(2) 50minute Incineration - temperature below 400C.jpg [ 116.39 KiB | Viewed 902 times ]
(3) Left - incinerated, scraped and treated with EDTA+SDS. Right - original treated chemically then rinswed with DW.jpg
(3) Left - incinerated, scraped and treated with EDTA+SDS. Right - original treated chemically then rinswed with DW.jpg [ 105.46 KiB | Viewed 902 times ]
(4) Incinerated and cleaned EDTA+SDS then spread in DW. 10X Neofluar.JPG
(4) Incinerated and cleaned EDTA+SDS then spread in DW. 10X Neofluar.JPG [ 259.04 KiB | Viewed 902 times ]
(5) Same, 40x Neofluar Ph2.JPG
(5) Same, 40x Neofluar Ph2.JPG [ 178.66 KiB | Viewed 902 times ]

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Last edited by Hobbyst46 on Sat Jul 14, 2018 8:53 pm, edited 5 times in total.
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PostPosted: Sat Jul 14, 2018 2:13 pm 
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(continued from above)


Attachments:
(6) Same, 40x Neofluar Ph2.JPG
(6) Same, 40x Neofluar Ph2.JPG [ 174.49 KiB | Viewed 901 times ]
(7) Same as 6.JPG
(7) Same as 6.JPG [ 174.01 KiB | Viewed 901 times ]
(8) Same as 6.JPG
(8) Same as 6.JPG [ 173.55 KiB | Viewed 901 times ]
(9) Same, 100x Planapo Oil Ph3.jpg
(9) Same, 100x Planapo Oil Ph3.jpg [ 36.77 KiB | Viewed 901 times ]
(10) Same as 9.jpg
(10) Same as 9.jpg [ 39.11 KiB | Viewed 901 times ]

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PostPosted: Sat Jul 14, 2018 2:14 pm 
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(continued from above. these are incinerated then chemically cleaned, except the bottom no 15 which is incinerated only)


Attachments:
(11) Same as 9.JPG
(11) Same as 9.JPG [ 152.36 KiB | Viewed 901 times ]
(12) Same as 9.JPG
(12) Same as 9.JPG [ 138.05 KiB | Viewed 901 times ]
(13) Same as 9.JPG
(13) Same as 9.JPG [ 196.83 KiB | Viewed 901 times ]
(14) Same as 9.JPG
(14) Same as 9.JPG [ 178.29 KiB | Viewed 901 times ]
(15) Incinerated only 25 min 40X.JPG
(15) Incinerated only 25 min 40X.JPG [ 178.66 KiB | Viewed 901 times ]

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Last edited by Hobbyst46 on Sat Jul 14, 2018 2:18 pm, edited 1 time in total.
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PostPosted: Sat Jul 14, 2018 2:16 pm 
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(continued from above. these are incinerated only)


Attachments:
(16)Same as 15.JPG
(16)Same as 15.JPG [ 284.88 KiB | Viewed 901 times ]
(17)Same as 15.JPG
(17)Same as 15.JPG [ 232.21 KiB | Viewed 901 times ]
(18)Same as 15.JPG
(18)Same as 15.JPG [ 82.75 KiB | Viewed 901 times ]
(19)Same as 15.JPG
(19)Same as 15.JPG [ 167.46 KiB | Viewed 901 times ]

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PostPosted: Sun Jul 15, 2018 1:21 am 
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Very nice work, Hobbyst46!

In that case, do you think incineration helped cleaning at all? In other words, if you clean the same sample without the first incineration step, would cleaning be less complete?

And the "incineration" we are doing here is different than that with a torch. Maybe torch would incinerate better and more evenly? I don't know if it will blow away diatoms though, if not controlled well. Maybe slightly higher temperature than 400C for a shorter time would actually work better than lower temp and longer time? I am only guessing.

Black powder does not sound as good as white powder. I have never obtained white power with my stove fire though.

Your incineration + chemical cleaning looks about as good as it can get with your mild reagents. I would add HCL abd 30% H2O2 after incineration, but I know/understand you want to avoid them.

The tiny mineral particles you have there may be very difficult to remove, without micromanipulation. They are probably only slightly denser than diatom frustules.


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PostPosted: Sun Jul 15, 2018 10:27 am 
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Thanks!
zzffnn wrote:
In that case, do you think incineration helped cleaning at all? In other words, if you clean the same sample without the first incineration step, would cleaning be less complete?
No, I think that incineration did not contribute significantly to the cleaning. But, your rational was that incineration will save most of the expensive chemical reagents used for further cleaning, and I think that this point may still be valid. In my opinion this goes as follows. The H2O2 you add is a volume of liquid and you add an excess, like 2:1 or 5:1 (or any other ratio) to the VOLUME of crude unclean diatoms. The excess is important, since you start with a strong reagent (30-35% as marketed) and you want to preserve the strength, not dilute it.
But that crude volume of diatoms and debris, although to the eye it is an opaque "concentrated" suspension, may actually contain just a small amount of solids. So, a relatively large volume of unwanted water.

Hence, to save on reagents, we should reduce our initial crude mass of diatoms and debris into as smaller volume, by removing the water. To do this, I see three options, and each means labor:

1. Rapid centrifugation. I think we object to it. Combined together, literature data and forum member opinions about it are quite inconclusive. So forget it.
2. Filtration through highly dense paper or membrane filter, under vacuum even. This is too time consuming and/or nuisance.
3. Heating to evaporate all the liquid. The idea just jumped into my mind, and is relatively simple to do.

A primary step of incineration of the mass, in fact, covers option 3 and beyond - get rid of redundant water as well as organic residues, so reduce the initial crude material to the absolute minimum volume before chemical treatment. Again, in my opinion, I think it is still worth testing, if one avoids the softening and deformation of the diatoms. So I hope to try it yet.

Quote:
And the "incineration" we are doing here is different than that with a torch. Maybe torch would incinerate better and more evenly? I don't know if it will blow away diatoms though, if not controlled well. Maybe slightly higher temperature than 400C for a shorter time would actually work better than lower temp and longer time? I am only guessing.
Yes, certainly different from what I did, like you say, a higher temperature and shorter duration. A torch may prove useful. However, I do not know how they applied the torch to the diatoms. My own experience (years ago) with directly heating coverslips in a gas burner or an alcohol flame was negative.The slips cracked, or spattered, or melted. The steel plate that I used above (the dimensions are borrowed from forum member MicroBob's protocol - thanks Bob!) prevents such problems, and at least, yields undamaged coverslips, and is fairly reproducible.
Using a torch on diatoms in a deep container, such as crucible or Pyrex bowl or beaker might work, but for me at least, it is too much trial and error (duration, distance from flame, container type etc).
BTW, a longer time (say double) and higher temperature (say by 10C) often yield the same result; not always. To melt a solid, one needs to heat to the melting point, and heating below melting point will not cause melting even if done through ages. But to burn an organic residue of a plant, time and temperature might be interchangeable.

Quote:
Black powder does not sound as good as white powder.
Perhaps this is not important, as long as it is a powder and not big lumps of tarry stuff.

Quote:
Your incineration + chemical cleaning looks about as good as it can get with your mild reagents. I would add HCL abd 30% H2O2 after incineration, but I know/understand you want to avoid them.
OK.

Quote:
The tiny mineral particles you have there may be very difficult to remove, without micromanipulation. They are probably only slightly denser than diatom frustules.
Agreed.
So, I plan to collect some fresh algae (without silt) and start again, at a lower temperature. I will do the same as above, only lower the stove flame to medium power. We shall see... and perhaps other people will find it interesting... :?:

P.S. The temperature on the coverslip is lower than the temperature on the metal plate, since glass, thin as it is, is an insulator. can be a difference of 30C and more. It does not change the conclusions though. In between the 1:0, 1:1 and 2:1 scores from Moscow, I managed to incinerate the black residue, created on the heated coverslip, some more on the bare plate. It remained mostly black, supporting the guess that it is mainly iron oxides.

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PostPosted: Mon Jul 16, 2018 9:42 am 
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The torch method doesn't work. Even the gas flame below a steel plate can blow diatoms away.

I too had the impression that diatoms can be deformed by incineration. So far I have no information that explains the reason. I had in Mind a melting point of about 1700 °C but this is obviously not the whole story as your results proove. It would really be nice to find a safe protocol for incineration
For diatoms from plancton incineration alone gives nice slides.

Bob


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PostPosted: Mon Jul 16, 2018 11:47 am 
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Hi Bob,
MicroBob wrote:
For diatoms from plancton incineration alone gives nice slides.
This makes sense, since the amount of organic matter inside the diatom and attached to the outside of the diatom is small, and the amount of insoluble ash that would remain after incineration of that organic matter might be so small that it would not be detected by light microscopy. I hope to find out whether incineration is effective for less clean "raw" samples, but restrict the experiments to heating on metal plates.

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PostPosted: Thu Aug 09, 2018 8:36 am 
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zzffnn wrote:
In that case, do you think incineration helped cleaning at all? In other words, if you clean the same sample without the first incineration step, would cleaning be less complete?

MicroBob wrote:
For diatoms from plancton incineration alone gives nice slides


Comparison of diatom cleaning protocols: oxidation, with vs without pre-incineration

In a previous post I reported on incineration of self-collected freshwater diatoms on coverslips, as an alternative to cleaning with chemicals, or an additional cleaning step. The results were not attractive, mainly because frustules were melted or at least deformed by the heat. Also, those raw samples were mostly silt, mud etc. Yet, the prospect of incineration as a pre-cleaning in order to destroy the organic matter as much as possible before the application of expensive chemicals is alluring. Incineration itself has seemingly occupied the minds of other MicrobeHunter Forum members as well. So, I thought I would incinerate a larger mass, and process the baked stuff with oxidant and acid.
Freshly collected, freshwater submerged decaying brown plants and algae, avoiding silt and mud, were wrung into water, and filtered through a 300 micron sieve, then divided into four identical portions and left to separate (natural gravitation only, no centrifuge). Thus each of the four test-tubes contained a volume of 3-4ml of dark brown precipitate, that was twice washed with DW (photo 1). Two of them, IncOx and Ox, respectively, were processed in the present run.
The contents of tube IncOx were transferred to a Pyrex 5cm diameter petri dish, and left to dry out. The dish was then placed directly on a wire mesh over the kitchen stove small flame and heated 30-40 minutes. This gave a dark brown mass (photo 2), volume about 0.5 ml. It was scraped with a spatula into a test-tube, to estimate its volume (photo 3), then poured into a beaker.
IncOx and Ox were each treated with 30% H2O2, at volume ratios of 1:8, for one hour in a boiling water bath. The precipitate color turned brighter. It was rinsed (back again in test tubes) several times with DW, then twice its volume of 31% HCl was added. After a few hours, final rinses with DW. The final colors are grey (Ox) and dark grey (IncOx) (photo 4). Further photos below depict the results.
Observations and conclusions (in italics):
1. Pre-incineration reduced the apparent volume of raw sample by a factor of ~5.
This will definitely help to save on further treatment chemicals. However, it is possible to reduce the volume in other ways than incineration. For example, filtration through filter paper or dense mesh, and centrifugation (the latter is somewhat controversial).
2. After both protocols, most of the diatoms were clean.
So, incineration has no advantage in this respect. Also, this demonstrates that the color of the product in the test-tube is not an unequivocal proof of cleanliness of the diatoms - a quite trivial conclusion!
3. The relative amount of non-diatom debris was somewhat higher after pre-incineration.
This is a disadvantage of incineration.
4. The debris after incineration was not charcoal.
So the temperature was sufficiently high for complete burn-off.
5. Incineration softened and deformed some of the frustules, although to a lesser degree than previous incinerations at higher temperatures.
This is another major disadvantage of incineration.

Based on these limited experiments, I cannot recommend incineration as alternative to chemical-based cleaning, nor as pre-treatment of a raw diatom sample, unless, perhaps, the diatoms are initially free of other stuff (perhaps freely swimming, net-collected diatoms).
The results are open to comments and criticism.


Attachments:
(1) IncOx - original precipitate from filtered collected sample.jpg
(1) IncOx - original precipitate from filtered collected sample.jpg [ 122.93 KiB | Viewed 694 times ]
(2) IncOx after incineration in petri dish.jpg
(2) IncOx after incineration in petri dish.jpg [ 155.36 KiB | Viewed 694 times ]
(3) IncOx after incineration, transferred from petri dish.jpg
(3) IncOx after incineration, transferred from petri dish.jpg [ 72.44 KiB | Viewed 694 times ]
(4) IncOx vs Ox after all treatments.jpg
(4) IncOx vs Ox after all treatments.jpg [ 120.64 KiB | Viewed 694 times ]

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Last edited by Hobbyst46 on Thu Aug 09, 2018 8:54 am, edited 2 times in total.
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PostPosted: Thu Aug 09, 2018 8:39 am 
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Here are the slide photos.

Note: a small mistake in the title of the photo with 40X objective - the 40X objective used is 40X0.75 Neofluar, not Plan.


P.S. I also tried boiling as pre-treatment - hope to report it in a later post.


Attachments:
IncOx mounted in air. BF, PLAN 25X0.45.jpg
IncOx mounted in air. BF, PLAN 25X0.45.jpg [ 292.01 KiB | Viewed 694 times ]
IncOx mounted in air. POL, PLAN 25X0.45.jpg
IncOx mounted in air. POL, PLAN 25X0.45.jpg [ 275.32 KiB | Viewed 694 times ]
Ox, mounted in air. BF, PLAN 25X0.45.JPG
Ox, mounted in air. BF, PLAN 25X0.45.JPG [ 294.13 KiB | Viewed 694 times ]
Ox, mounted in air. Oblique, PLAN 40X0.75.JPG
Ox, mounted in air. Oblique, PLAN 40X0.75.JPG [ 250.28 KiB | Viewed 694 times ]
Ox, mounted in air. BF+POL, PLAN 25X0.45.JPG
Ox, mounted in air. BF+POL, PLAN 25X0.45.JPG [ 221.77 KiB | Viewed 694 times ]

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PostPosted: Thu Aug 09, 2018 1:59 pm 
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Hobbyst46,

Thank you for the beautiful comparison! Your results are consistent and you have convinced me that incineration does not help, in our setting.

Have you determined how many hours of chemical treatment is necessary for cleaning?

I seemed to see lighter color after 48 hours of 31% HCL, compared to 24 hrs (but I have not compared images and lighter color may not mean better cleaning). My experiment was with 20ml of dirty precipitates (much higher starting volume) and 31% HCL after 30% H2O2 though. I did long incubation, because I thought (perhaps mistakenly), since human stomach need a few hours of HCL and shear force to digest plant matters, my incubation may need longer, as it does not have shear force.


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PostPosted: Thu Aug 09, 2018 4:47 pm 
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zzffnn wrote:
Have you determined how many hours of chemical treatment is necessary for cleaning?
Thanks for the appreciation.
I would like to answer by providing some more details. I treated IncOx and Ox each with 30% H2O2 in a beaker, at volume ratios of 1:8, for one hour in a boiling water bath. The precipitate color turned brighter than before, but remained grey-brown. I then transferred it to glass test tubes, and rinsed several times with DW. The solids separated within 30-60 minutes. It is very important to eliminate all H2O2 by several rinses before the addition of acid. In the early evening I removed most of the liquid above the precipitate, and added twice the volume of the solids of 31% HCl. Within a few hours the color brightened further. I left it at room temperature overnight. Next morning, I began rinsing with DW. The final colors are grey (Ox) and dark grey (IncOx). The diatoms were essentially clean, although occasionally, some lipid spherules were seen on or within otherwise clean frustules. I took pictures again, after 4-5 days of further rinsing (last two rounds with ethanol) and all diatoms looked clean.
Initially, organic matter resides both inside the live diatom and on its external surface; diffusion of the decomposed stuff out from within the frustule could be much slower than removal from the external surface ; nevertheless, shear forces could perhaps prevent blockage of the pores and openings in the frustule. The cleaning is IMO a complex mechanism. I think that 24 hours of acid treatment should suffice to clean almost all diatoms, and the further rinses will make it complete. To reach a pH of >5, starting with 20-30% HCl, requires dilution of 1:1,000,000, or six 10X consecutive dilutions, another day's job.
So, I guess 1 hour hot H2O2, 20 hours RT HCl, plus the rinse time.
Quote:
I seemed to see lighter color after 48 hours of 31% HCL, compared to 24 hrs (but I have not compared images and lighter color may not mean better cleaning). My experiment was with 20ml of dirty precipitates (much higher starting volume) and 31% HCL after 30% H2O2 though. I did long incubation, because I thought (perhaps mistakenly), since human stomach need a few hours of HCL and shear force to digest plant matters, my incubation may need longer, as it does not have shear force.
I fully agree with your reasoning. Boiling with HCl (like Charles and others have processed their diatoms) can definitely shorten the time schedule, maybe by a 1-2 order of magnitude, by providing both energy bubble shear force, but I personally rejected it. As I mentioned above, IMO, the correlation between the color of the processed mass and the cleanliness of the frustules is partial.

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PostPosted: Thu Aug 09, 2018 5:25 pm 
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Why is it important to remove all H2O2 before addition of acid? H2O2 is usually stored under acidic condition.

And is it necessary to heat 30% H2O2 solution? Perhaps it is safer to let reaction go on at room temperature?


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PostPosted: Thu Aug 09, 2018 7:12 pm 
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zzffnn wrote:
Why is it important to remove all H2O2 before addition of acid? H2O2 is usually stored under acidic condition.

Yes, H2O2 is more stable under acidic conditions than under base. However, H2O2 and HCl are known to react with each other. I inadvertently introduced 31% HCl into a diatom mass right after 30% H2O2 treatment, without proper rinse in between. A very intensive heating and bubbling resulted and appeared to go out of control. The colorless liquid yellowed (free chlorine?). Perhaps the diatom mass catalyzed something (?). So, while I am not sure about what happened, I would suggest to use them separately.
Incidentally, a Brazilian paper from year 2014 states that the optimal pH, for bleaching of some wine and tobacco pigments is 9 (when the starting pH is 6 and up!).

Quote:
And is it necessary to heat 30% H2O2 solution? Perhaps it is safer to let reaction go on at room temperature?
I agree that 30% H2O2 is safer when cold. I performed the heating by immersing the beaker/conical flask with diatoms and H2O2 in boiling water within a larger beaker. Bubbling was stable, though intense. How long will the cleaning take at RT? I do not know. Worth a try...

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PostPosted: Thu Aug 09, 2018 9:32 pm 
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Yes, I totally forgot about it. I also saw/smell yellow chlorine as well as heating and bubbling (it was not terrible, but I saw it for sure). It does make sense, when we look at the reaction mixture and chemistry equation.


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PostPosted: Fri Aug 10, 2018 10:32 am 
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zzffnn wrote:
Bubbling action of H2O2 also helps, so it is not just oxidation. Steve Beats (diatomist) of UK said gentle boiling / bubbling of water itself can clean quite some fresh diatoms. Use H2O2 last, because 30% H2O2 is not cheap....

As mentioned above, I had prepared freshly collected, freshwater submerged decaying brown plants and algae, avoiding silt and mud, wrung into water, filtered through a 300 micron sieve, and rinsed twice with DW.

To learn some about boiling, I transferred a portion of that raw sample, containing 3-4ml of dark-brown fluffy precipitate, into a Pyrex test-tube, and immersed the loosely-covered tube in a pot of boiling water for 0.5 hour. The fluffiness disappeared, and the color changed into near-black. The solids were rinsed with DW, then treated twice, repeatedly, with a double the volume of 3% H2O2 (not 30%. 30% would definitely work, but I wanted 3%). The color lightened to brown.
Inspection of the processed diatoms showed partial cleaning, in accord with Steve Beats, in principle (see photos 1-2). But I did not like the result. So I added half the volume of a laundry stain remover, a viscous liquid named "Vanish", and left it overnight. Then, after rinses with DW, most of the diatoms were found to be mostly clean (see photos 3-4). There is a lot of other debris, though, worse than after other cleanings.
Within the limited framework of these experiments, boiling in water contributes some cleaning, but must be followed by treatment with chemicals.

I am still tempted to subject raw diatom samples to "laundry" - boiling them straight ahead with Vanish, or Tide. Has anyone tried it ?


Attachments:
(1) Boiled, treated with 3% H2O2 (2X).JPG
(1) Boiled, treated with 3% H2O2 (2X).JPG [ 238.92 KiB | Viewed 633 times ]
(2) Boiled, treated with 3% H2O2 (2X).JPG
(2) Boiled, treated with 3% H2O2 (2X).JPG [ 278.57 KiB | Viewed 633 times ]
(3) Boiled, treated with 3% H2O2 (2X) then Vanish.JPG
(3) Boiled, treated with 3% H2O2 (2X) then Vanish.JPG [ 261.29 KiB | Viewed 633 times ]
(4) Boiled, treated with 3% H2O2 (2X) then Vanish.JPG
(4) Boiled, treated with 3% H2O2 (2X) then Vanish.JPG [ 296.09 KiB | Viewed 633 times ]

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PostPosted: Fri Aug 10, 2018 5:37 pm 
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Laundry detergent overnight may be too harsh, as its PH value is high (may eat away frustules). Maybe shorten treatment to 3 hrs? Laundry bleach protocol says no more than 4 hrs, if I remember correctly.

Thank you for your hard work!


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PostPosted: Fri Aug 10, 2018 7:46 pm 
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zzffnn wrote:
Laundry detergent overnight may be too harsh, as its PH value is high (may eat away frustules). Maybe shorten treatment to 3 hrs? Laundry bleach protocol says no more than 4 hrs, if I remember correctly.

Thank you for your hard work!
Great to share the brain storming! but it is more a pleasure than hard work, with the tiny prospect of "discovery".
Regarding the pH and duration of the treatment. Indeed, I avoid hypochloric bleach in this case. So, pulled two stain removers off my (home) shelf: "Vanish Gold" - by Reckitt, and "Tide with Bleach Alternative" by Procter&Gamble. According to the directions on the package, both are appropriate for color clothing, and there is no laundry time limitation, and none of them contains hypo. I prepared concentrated aqueous solutions, checked the pH with 4-color indicator paper strips, found it to be 7.0-7.5 for both, and the indicator strip colors did not fade. So these are very gentle oxidants (hopefully, not too gentle).
I will get me fresh diatoms and run 3 tests: (a) RT 30% H2O2, (b) Boil in Vanish, (c) Boil in Tide. And check the diatom cleanliness after 3h and after a longer period.

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PostPosted: Fri Aug 10, 2018 8:36 pm 
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Sounds good. Sorry for my mistaken speculation on pH of your laundry detergent. Perhaps many of those gentle laundry products are pH neutral? For pH7, you don't have to shorten treatment to 3hrs.


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PostPosted: Sat Aug 11, 2018 3:40 pm 
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Actually, I also have a laundry brightener powder (from the supermarket), that is based on sodium percarbonate, hence it releases H2O2 when dissolved in water. I do not know the concentration, but the pH is basic: 10-11. Hence, it is somewhat less basic than chlorine bleach. Might be interesting to include it in the test, with too differences vs the other agents: RT (not boil) and short term treatment.

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PostPosted: Sun Aug 12, 2018 8:01 pm 
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Unfortunately, the last cleaning diatom adventure, described below, did not bring anything new.
Like previously, I have prepared 4 identical samples of raw freshwater epiphytic diatoms. Each sample was treated differently:
zzffnn wrote:
And is it necessary to heat 30% H2O2 solution? Perhaps it is safer to let reaction go on at room temperature?


1. 30% H2O2 at room temperature (28-30C - the heat of Jan-Feb-Mar-Apr-May-Jun-Jul-Aug 2018 is record breaking)). Actually, 17ml H2O2 was added to a
3ml sample.
Checked after 3-4 hours and again after 8 hours. Although the initial very dark brown color turned bright brown, the diatoms were only slightly
deprived of their organic contents. So, after 8 hours, I immersed the test-tube in warm water (70C and declining to about 50 in 20 minutes). Better
cleaning of the diatoms resulted. Importantly, non-diatom debris has been mostly destroyed.


2. One hour boiling with the detergent Tide.
The color of the mass turned black and it is less fluffy and floating, but almost no cleaning of the diatoms, and the non-debris is as bad as before
cleaning, Three hours after start. There was no release of bubbles before or after the boiling.


3. One hour boiling with the detergent Vanish.
The color of the mass turned somewhat brighter brown, but otherwise the results are as poor as with Tide, despite the intensive release of oxygen
bubbles during the first hour. Three hours after start.


4. Three-four hours RT incubation with Ran, a laundry additive based on percarbonate (Na2CO3.H2O2) that has a pH of 10-11.
Astonishingly, results are as poor as with the other detergents, despite the intensive release of oxygen bubbles during the first hour.

It appears that:
(a) H2O2 is really more effective when the mixture is warmed. RT treatment is not completely effective even after 8 hours.
(b) Those detergents are not effective, at least when used alone, since even if the diatoms would eventually become become cleaner, non-diatom debris remains plentiful.
I think I will repeat the H2O2 experiment, using a higher excess of H2O2, and wait 24h at RT, just to make sure.

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PostPosted: Tue Aug 14, 2018 9:03 am 
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zzffnn wrote:
I seemed to see lighter color after 48 hours of 31% HCL, compared to 24 hrs (but I have not compared images and lighter color may not mean better cleaning). My experiment was with 20ml of dirty precipitates (much higher starting volume) and 31% HCL after 30% H2O2 though. I did long incubation, because I thought (perhaps mistakenly), since human stomach need a few hours of HCL and shear force to digest plant matters, my incubation may need longer, as it does not have shear force.
I have done some more experiments that all together convey the following information. It reconfirms statements and lessons already mentioned in protocols, the research literature, and posts in this respectable forum. So it is just another demonstration, with another raw environmental diatom sample.

1. H2O2 30% (or close it, depending on the ratio of diatom volume to H2O2 volume) works much much better when the tube (or flask or beaker) is immersed in a boiling water bath, than at room temperature. One hour at boiling is better than 24 hours at RT. In the RT sample, diatoms were barely cleaned. After boiling, they are mostly clean (though not absolutely).
Since the bubbles this case are mostly oxygen (and some CO2) rather than noxious fumes, the only danger is overflow of the liquid outside from the container. So, place the mass to be digested in a much larger flask.

2. There is a weak correlation between the color of the raw diatom sample (that contains non-diatom debris) and the cleanliness of the diatoms themselves. In the example above, part of the sample digested with H2O2 at RT was treated with HCl after 12 hours: the color of the solids turned from brown to light grey within minutes, but the diatoms were not significantly cleaner (after 2 hours). Another example: the sample digested with H2O2 at RT stayed light brown after 24 hours, and diatoms were not clean; after an hour of boiling, the color stayed the same, but diatoms were mostly clean.
That brightening of the color of the treated mass is not a reliable indicator of diatom cleanliness is quite trivial, since the color originates from non-diatom debris as well. IMHO, still, it is a nice demonstration.

3. HCl acts slowly at RT. After two hours, at ~15% HCl (31% HCl added on a 1:1 volume ratio), no significant cleaning (following the 12 hours RT digestion with H2O2). Boiling with HCl will probably be much more effective, but in contrast to H2O2, boiling HCl gives off noxious corrosive and dangerous fumes (HCl) so - never for home use, requires professional expertise!.

Some more technical details:

The raw sample of diatoms and plant debris, with barely any silt and mud, was wrung into water, filtered through a 300 micron sieve, and rinsed twice with DW. It formed 3-4ml of dark-brown mass of flocculate in a test-tube. I added 15-20 ml of H2O2 30% to digest it at RT (28-30C). Designated H2O2-RT. Very slow bubbling occurred and the overall color of the liquid + solid turned slowly to medium grey-brown. However, after 5 and 8 hours (photos) the diatoms themselves were only slightly cleaned, or perhaps ripped of their external organic slime - but the inside contained a lot of organic stuff - yellow/green pigments. After 12 hours, a small portion of the sample was drawn into a separate tube and drops of HCl 31% were added (so final conc of HCl about 15%). Call it sample H2O2-RT-HCl. It was left at RT for 12 additional hours, and its color turned light-grey, yet the diatoms still contained a lot of organic stuff - yellow/green pigments.

So, H2O2 ~25% at RT for 12 hours, plus ~15% HCl at RT for another 12 hours was not efficient at RT.

Sample H2O2-RT did not change even after 24 hours: the overall color of the solid mass was light brown, and the diatoms still contained a lot of organic stuff - yellow/green pigments (photos). I transferred it into Pyrex test-tubes, and immersed the open tubes in a pot of boiling water for one hour. Strong effervescence started and persisted. Upon cooling, the bubbling stopped. The solids regained their previous light brown color, but most of the diatoms were fairly clean, although some organic residues remained.

So, digestion with H2O2 is better done in a boiling water bath, for 1 hour.

And - the color of the solid diatom-debris precipitate is not a reliable indicator of the cleanliness of the diatom frustules themselves.


Attachments:
5 hours H2O2 RT. Most medium diatoms and tiny diatoms contain pigments (1). Two gas bubbles (probably oxygen) are visible.JPG
5 hours H2O2 RT. Most medium diatoms and tiny diatoms contain pigments (1). Two gas bubbles (probably oxygen) are visible.JPG [ 186.49 KiB | Viewed 528 times ]
5 hours H2O2 RT. Most medium diatoms and tiny diatoms contain pigments (3).JPG
5 hours H2O2 RT. Most medium diatoms and tiny diatoms contain pigments (3).JPG [ 183.11 KiB | Viewed 528 times ]
8 hours H2O2 RT. Medium diatoms still contain pigments(1).JPG
8 hours H2O2 RT. Medium diatoms still contain pigments(1).JPG [ 155.55 KiB | Viewed 528 times ]
8 hours H2O2 RT. Medium diatoms still contain pigments(2).JPG
8 hours H2O2 RT. Medium diatoms still contain pigments(2).JPG [ 185.87 KiB | Viewed 528 times ]
8 hours H2O2 RT. Medium diatoms still contain pigments(3).JPG
8 hours H2O2 RT. Medium diatoms still contain pigments(3).JPG [ 189.39 KiB | Viewed 528 times ]

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PostPosted: Tue Aug 14, 2018 3:43 pm 
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More photos of the samples in the test tubes and the results.


Attachments:
Left- raw collected mass, after filtration and rinse with DW. Right - after 5 hour of digestion with H2O2 30%, RT.jpg
Left- raw collected mass, after filtration and rinse with DW. Right - after 5 hour of digestion with H2O2 30%, RT.jpg [ 138.64 KiB | Viewed 528 times ]
H2O2 30% RT 12h then HCl RT 12h. Color of the solid precipitate is light grey, yet diatoms are not clean.jpg
H2O2 30% RT 12h then HCl RT 12h. Color of the solid precipitate is light grey, yet diatoms are not clean.jpg [ 94.41 KiB | Viewed 528 times ]
24 hours H2O2 RT. Diatoms still contain pigments(4). Gas (oxygen) bubbles are visible.JPG
24 hours H2O2 RT. Diatoms still contain pigments(4). Gas (oxygen) bubbles are visible.JPG [ 200.76 KiB | Viewed 528 times ]
24 hours H2O2 RT. Diatoms still contain pigments(5). Gas (oxygen) bubbles are visible.JPG
24 hours H2O2 RT. Diatoms still contain pigments(5). Gas (oxygen) bubbles are visible.JPG [ 172 KiB | Viewed 528 times ]
24 hours H2O2 RT. Diatoms still contain pigments(7).JPG
24 hours H2O2 RT. Diatoms still contain pigments(7).JPG [ 137.93 KiB | Viewed 528 times ]

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PostPosted: Tue Aug 14, 2018 3:45 pm 
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Here are some photos of the diatoms after 24 hours of RT digestion with H2O, THEN 1 hour boiling. More results due within 1-2 days.


Attachments:
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (8).JPG
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (8).JPG [ 147.95 KiB | Viewed 528 times ]
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (9).JPG
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (9).JPG [ 141.71 KiB | Viewed 528 times ]
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (10).JPG
24 hours H2O2 RT then 1 hour boiling. Diatoms are mostly clean (10).JPG [ 154.49 KiB | Viewed 528 times ]

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PostPosted: Tue Aug 14, 2018 3:53 pm 
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^ Very nice work, thank you! Your findings will surely help me and other hobbyists. I agree with you and will boiling H2O2 on large container.


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PostPosted: Tue Aug 14, 2018 4:09 pm 
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Just the (notorious) safety note - I always wear safety goggles for boiling ANYTHING. I wear rubber gloves for handling H2O2 30%, and I bring the digestion to boil gradually, under supervision.

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