More on mild cleaning and incineration of diatoms

[Hobbyst46]

This post is a continuation of the thread:
viewtopic.php?f=10&t=6032&start=30

Cleaning diatoms by incineration.

I expected that incineration will eliminate the organic constituents of the diatom, even without chemical treatment. In addition, I wondered whether incineration will attach ("weld") diatoms to the cover-slip, as I have read somewhere. I planned to work on epiphytic diatoms, to avoid silt, sand etc. However, my only available live diatoms were on algae from a tiny artificial freshwater pond, containing very few genera. I tried incineration as well as mild cleaning with EDTA+detergent, followed by hot 3% H2O2 (common pharmacy H2O2) as detailed in the previous post thread. All rinses and liquid changes were based on gravitational settling of the solids, without centrifuge, and supernatant liquid withdrawal with a syringe or pipette.
For incineration, a drop of diatom suspension was left to dry out on a 18x18 coverslip. Then the slip was placed flat on a 2mm thick rectangular (~57x33mm) plate. The plate was laid on a stainless steel wire mesh, over the small kitchen stove gas flame of diameter ~7cm. Within less than 10 minutes, the temperature of the top surface reached 460-490C. Total heating time was 20-25 minutes. The slips remained flat and intact, without any fractures.

Samples were mounted in polystyrene-cinnamon bark oil, and sealed with paraffin by using a heated L-shaped brass rod to melt the wax and coat the slide along the edge of the coverslip (photo).

Pressing and squeezing the algae into water, filtration through a 0.6mm mesh and settling in test tubes yielded the dark brown solids (photo A, "start"). Several rinses with distilled water, followed by incineration, yielded clean diatom frustules, mixed with many other grey-white particles ("rinse" - PD2').
Photos: brightfield - either 10x0.30 or 16x0.40 (Neofluars); darkfield - 40x~0.8 Planapo (oil; oiled condenser); phase contrast - 63x1.4 PH3 Planapo (oil). Diatoms are mounted in PS-CBO.

Results (some of them are demonstrated by the photos).

1. The dominant large diatoms were 200-400micron long pennates, which I think are Ulnaria (might be wrong). Many of them are broken, possibly from the sampling step.
2. Incineration of the thoroughly rinsed sample alone cleaned the diatoms, yet non-diatom debris also remained. Chains of diatoms did not break into individuals. Ulnaria forms were similar to live Ulnarias.
3. Cleaning with EDTA+SDS, then 3% H2O2 cleaned most of the diatoms . Incineration afterwards cleaned them all. Non-diatom debris was less abundant, relative to the amount of diatoms, than in incinerated-only samples. Ulnarias were separated into valves (please correct if I'm wrong).
I believe that the dark color of the solids in the test tubes after cleaning is due to non-diatom debris, although it could also result from incompletely clean frustules.
4. The frustules of small (<50micron) triangular diatoms, that had been destroyed completely in a previous cleaning project, that included boiling in acids and 30% H2O2 and centrifugations), remained unharmed after the mild cleaning and/or incineration.
5. Incineration softened some of the frustules and lead to bending and deformation of Ulnaria as well as some very small (navicula?) boat-like frustules.
6. Despite the softening, incineration did not "weld" the diatoms to the coverslip. Even slight pressure on the coverslip of the prepared slide pushed frustules away and out of the field of view.

Hopefully these data will be found useful for others who are interested in incineration of diatoms.

[Hobbyst46]

Here are some mild chemical cleaning +incineration result demonstrations.

[Hobbyst46]

Here are some incineration-only results demonstrations

[MicroBob]

Hi Doron,
thank you for sharing this detailed report with us. For me at least it is a very interesting starting point for further experimentation.
Here some comments from me:
- The remaining debris after incineration: I would expect some debris as there must at least be the ashes from the incineration. I'm not sure how long perfect incineration takes at this temperature. May be the oxygen from the surrounding air does not reach into thicker layers of debris.

- The deformed diatoms: The diatom material is not too far from glass. Glass moves at room temperature, but very slow. It starts to move quicker at temperature around 500 °C. How did you measure the temperature? If your measurement was right, 460 to 490 °C would be already too high for some diatoms at this duration.

- Diatoms not sticking: Incineration is especially interesting for diatom chains. Whith chemival cleaning these are scattered and difficult to identify. From my experiments I can say that the slides were not especially pretty but the diatoms were clean and the chains intact. Overall perfectly useable slides.

- Diatoms surviving incineration better than chemical cleaning: I know that both types of cleaning can harm some diatoms, but I would have expected that incineration is overall more harmful. Diatoms are really a difficult topic!


I will soon go on holiday to near Dresden here in Germany. I will try to collect some diatoms too.

[Hobbyst46]

Hi Bob, and thanks for the comments!
Some of my experiments were motivated by your protocol!

- The remaining debris after incineration: I would expect some debris as there must at least be the ashes from the incineration. I'm not sure how long perfect incineration takes at this temperature. May be the oxygen from the surrounding air does not reach into thicker layers of debris

Yes, I agree. The only point I can add is that after about 10 minutes at 460-480 but even at 400, no black spots were seen on the slide. So burnout was complete. Ashes usually melt and vaporize at much higher temperatures, say 900-1000 or more, so they would remain as white-gray traces.

- The deformed diatoms: The diatom material is not too far from glass. Glass moves at room temperature, but very slow. It starts to move quicker at temperature around 500 °C. How did you measure the temperature? If your measurement was right, 460 to 490 °C would be already too high for some diatoms at this duration.

This is apparently the case. I measured the temperature with a high quality digital thermometer. The sensor was a type K thermocouple, which I calibrated beforehand. To measure during incineration, I touched the sensor tip to the top surface of the steel plate and waited until a more or less stable reading.

- Diatoms not sticking: Incineration is especially interesting for diatom chains. Whith chemival cleaning these are scattered and difficult to identify. From my experiments I can say that the slides were not especially pretty but the diatoms were clean and the chains intact. Overall perfectly useable slides.

I agree. In my experiments, diatom chains were preserved, diatom pairs and threesomes ( ) were intact, and needle-like diatoms (Syndera I think they are) appear in girdle view rather than in valve view.
What I thought about "sticking" was the attachement of diatoms to the glass - by mutual softening, sort of. That did not happen.

- Diatoms surviving incineration better than chemical cleaning: I know that both types of cleaning can harm some diatoms, but I would have expected that incineration is overall more harmful. Diatoms are really a difficult topic!


I will soon go on holiday to near Dresden here in Germany. I will try to collect some diatoms too.

Enjoy! heard much about the beauty of Dresden and the natural reserves nearby.

[zzffnn]

Doron,

I cannot believe I forgot to congratulate you on your very nice work. Sorry about that.

Your cleaned diatoms look very close to those cleaned with harsh chemicals. Sample source variation plays a big part in cleaning results, so your cleaning may be as good, if not better.

I am guessing most kitchen stove fire can achieve 400C or more, if not 500C. So your results will help many hobbyists. Do you think 400C or 450C would work just as well (sorry, I have not followed up on incineration enough to remember or know it).

Also, have you tried incineration first, then 3% H2O2, then SDS + EDTA? That arrangement may reduce material bulk, increase cleaning speed and reduce reagent cost.

Thank you!

I read the two papers you cited for EDTA+SDS, as well as reviewing my previous lab notes. Indeed, SDS + EDTA gently disrupts cell membrane, extracts/denatures proteins and lipid; those actions clean diatom. Those reagents are also edible (and are very safe), to some extent. But they won't clean diatoms completely, without help of H2O2 hydrogen peroxide. For example, I doubt polysaccharides (plant fibers) are sufficiently removed/destroyed by SDS+EDTA.

One of those papers also mentioned that 30% H2O2 may destroy some very fragile frustules, by destroying some supporting molecules in diatom cells. 3% hydrogen peroxide may be much gentler, though may be less effective.

[MicroBob]

I measured the temperature with a high quality digital thermometer. The sensor was a type K thermocouple, which I calibrated beforehand. To measure during incineration, I touched the sensor tip to the top surface of the steel plate and waited until a more or less stable reading.
I can't see much cause for errors here so we can safely assume that 460-490 degrees Celsius for 20 minutes are already too much. This quite a useful bit of information! Glass is not melting like ice but its becoming increasingly soft over a wide range of temperatures. I guess that 30 degrees less will already improve things.

- Diatoms not sticking: Incineration is especially interesting for diatom chains. Whith chemival cleaning these are scattered and difficult to identify. From my experiments I can say that the slides were not especially pretty but the diatoms were clean and the chains intact. Overall perfectly useable slides.

I agree. In my experiments, diatom chains were preserved, diatom pairs and threesomes ( ) were intact, and needle-like diatoms (Syndera I think they are) appear in girdle view rather than in valve view.
What I thought about "sticking" was the attachement of diatoms to the glass - by mutual softening, sort of. That did not happen.

My diatoms and dirt stuck to the coverslip enough to keep them from sinking down into the mountant while I'm making the slide. So they are all in one focus plane and ageeeable to look at. Probably they are held to the cover slip by burnt remnants of the outer coating of the diatoms. My impression is that valves are mostly not separated by incineration without an additional chemical step afterwards. This often offers a very different look at well known diatom species.

[Hobbyst46]

Your cleaned diatoms look very close to those cleaned with harsh chemicals. Sample source variation plays a big part in cleaning results, so your cleaning may be as good, if not better.

Yes, for initially epiphytic diatoms.

I am guessing most kitchen stove fire can achieve 400C or more, if not 500C. So your results will help many hobbyists. Do you think 400C or 450C would work just as well (sorry, I have not followed up on incineration enough to remember or know it).

Yes, I believe even 350 would be fine. I know that below 300 left dark spots, i.e. charcoal instead of ash. I will try to repeat incineration at 400 and below, will take a couple of weeks since I need another steel plate and a fresh sample of live epiphytic diatoms.

Also, have you tried incineration first, then 3% H2O2, then SDS + EDTA?

I will answer a little later.

I read the two papers you cited for EDTA+SDS, as well as reviewing my previous lab notes. Indeed, SDS + EDTA gently disrupts cell membrane, extracts/denatures proteins and lipid; those actions clean diatom....But they won't clean diatoms completely, without help of H2O2 hydrogen peroxide. For example, I doubt polysaccharides (plant fibers) are sufficiently removed/destroyed by SDS+EDTA.

Agreed. There is some compromise on cleanliness here - SEM microscopists will probably reject my gently cleaned diatoms...

One of those papers also mentioned that 30% H2O2 may destroy some very fragile frustules, by destroying some supporting molecules in diatom cells. 3% hydrogen peroxide may be much gentler, though may be less effective.

Yes, more on that a little later.

Thanks for the compliments!!

[zzffnn]

Doron,

I read your SDS+EDTA protocol and compared it to that of the original paper. I think you did the best you could, without access to lab grade votex machine.

Original paper says vortex 6 times, which is what is usually done with SDS in a lab (SDS needs to foam a lot to work as a detergent to break up cells). For your protocol, I suggest increasing cleaning solution volume relative to diatom sample volume (by removing excess water and use just precipitate). And hand shake the SDS solution as much as you can during your 6 hr incubation.

Your protocol works as it is; the above change may make it clean even better.

Cheers,
Fan

[Hobbyst46]

... we can safely assume that 460-490 degrees Celsius for 20 minutes are already too much... I guess that 30 degrees less will already improve things.

Agreed. I will try it, only that I have to find a new steel plate, the previous one is too small.

My diatoms and dirt stuck to the coverslip enough to keep them from sinking down into the mountant while I'm making the slide. So they are all in one focus plane and ageeeable to look at. Probably they are held to the cover slip by burnt remnants of the outer coating of the diatoms. My impression is that valves are mostly not separated by incineration without an additional chemical step afterwards. This often offers a very different look at well known diatom species.

My incinerated diatoms were swept away by liquid mountant (CBO- cinnamon bark oil). But, using PS-CBO, they are OK even for the 100X oil immersion obective.

@MicroBob
@zzffnn
First, "flame temperature" is difficult to measure, since flame structure is quite complex. We actually measure the temperature of the heated object, which is a solid body or a volume of liquid in a closed/open container or a gas in closed container. To measure the temperature (T) of the incinerated diatoms, I actually measure the surface of a plate in contact with cover slips. Since the metal is heat conducting and the slip is very thin, I ASSUME that the diatoms on the slips reach T upon heating.
I am saying this because the incineration protocol I used, and the obtained temperatures, are specific to a small amount of DRY solid material on a stainless steel plate of the size I mentioned. I assume, that if one places a small amount of live or fixed diatoms (and the accompanying debris) on the plate on a similar stove flame, it will reach the same T within the same speed.

Having never tried incineration prior to cleaning, I try to imagine how I would do at least the incineration step for this purpose.

Obtain the raw uncleaned diatom precipitate (flocculate), say a volume of 1ml in the test tube. Assume its solid dry content is small - say, 200-300mg. Filter it through a filter paper (ashless filter paper like used in elementary chemistry labs, not home "tissue" stuff) circle to remove water. Let the paper circle dry out completely. Fold the paper piece into a small blob that contains the dry stuff, and place it on a large cover-slip - 60x24mm, 40x24mm etc. Put that on the steel plate and over the flame, to burn. Expect fly ash, some smoke, spouse in panic, Fireman Sam rushing in etc . When the black residue becomes gray-white, it should remain over the flame for 10-15 minutes to incinerate more. Leave it to cool down. With a soft brush, remove the residue into a small test tube for further cleaning with any chemical you like. Or wash it with DW, if it is a powder.

Will chemicals be effective on the incinerated mass? HCl, or warm HCl, is likely to dissolve at least some of it. H2O2 30% - no, since there is no organic residue left. EDTA- perhaps somewhat, I cannot tell. SDS - can help break the solid residue into small particles, I doubt if that is important.

So, the "incineration first, chemicals second" is doable, but I somehow doubt its success.

At any rate, the stainless steel size should be larger than what I used. I suggest 90x33mm, 2 thickness.
I will try to ASAP get a new plate, larger than my previous one, re-verify temperatures, and when I can collect a fresh sample of diatoms, test at least the incineration step.

In fact, I tried a harsher treatment to the gently cleaned diatoms - more on that, and a photo, later.

[zzffnn]

Hobbyst46,

Incineration should theoretically destroy all organic materials and clean diatoms by itself. But as you can see from actual results, it may not be complete and gentle at the same time. But using it first seems plausible, because it is effective at reducing bulk and cheap.

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 and you really don't need that much, after incineration (if hou use it before incineration, you may need much more H2O2).

After incineration, HCL + H2O2 should be more effective for cleaning diatoms (again, you don't need much after incineration). I mentioned SDS + EDTA, just in case people don't want to use strong evaporating acid. SDS+EDTA may remove some metal ions, but may not be as effective as HCL.

[Hobbyst46]

Hobbyst46,

Incineration should theoretically destroy all organic materials and clean diatoms by itself. But as you can see from actual results, it may not be complete and gentle at the same time. But using it first seems plausible, because it is effective at reducing bulk and cheap.

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 and you really don't need that much, after incineration (if hou use it before incineration, you may need much more H2O2).

After incineration, HCL + H2O2 should be more effective for cleaning diatoms (again, you don't need much after incineration). I mentioned SDS + EDTA, just in case people don't want to use strong evaporating acid. SDS+EDTA may remove some metal ions, but may not be as effective as HCL.

I fully accept the rationale. I also see two targets here - to destroy and eliminate the organic part of the diatom itself, and to destroy and eliminate non-diatom stuff. What I am concerned about is the physical state of the post-incineration residue - how accessible will it be to the chemicals. We do not want to use intense mixing, pulverization, ultrasonics to break the solid lumps. But, maybe my concern is not realistic.

At any rate, I am planning to run at least the incineration step on a mass of diatoms (rather than on dried drops of suspension on cover-slips as before) and see.
BTW: My cleanest batch of diatoms, after the EDTA+SDS+3% H2O2 cleaning, was still of a gray-brown color. I treated it with 30% H2O2, in a warm water bath (no boiling), and it turned white. Microscopical observation showed that non-diatoms debris has been eliminated, and the proportion of completely cleaned diatoms increased. However, certain diatoms (the nicest forms of course) broke apart.

[Hobbyst46]

Doron,

I read your SDS+EDTA protocol and compared it to that of the original paper...For your protocol, I suggest increasing cleaning solution volume relative to diatom sample volume (by removing excess water and use just precipitate). And hand shake the SDS solution as much as you can during your 6 hr incubation.

About the volume ratio - agreed. Mine was less than favorable, which is why I had to repeat the treatment. Vortex mixing - is certainly benefical, but will it break fragile frustules? Hand shake - yes, gently...
BTW, SDS introduced a lot of foam and bubbles, though not as much as 30% H2O2.

Thanks for all comments!

[MicroBob]

I once had assembled a collection of smelly diatom samples that awaited cleaning . To speed up the process I incinerated them directly on a big stainless steel plate of about 12x12cm in my oven. The samples had quite some volume and I had to scrape the sample of the plate with a sharp knife. Together with the sample I removed quite a bit of dark grey scale from the stainless steel itself.
So I would not advise to put the sample directly onto a piece of steel. A ceramic tile might be interesting as long as it is robust enough. What is used in a laboratory to heat samples up to 450°C? Special glass ware or porcelain? When using a deeper vessel, it could be possible to use it throughout the cleaning steps.

I think that chemical cleaning should be easier after incineration than before. I would expect ashes to be powdery and alcalic, easy to get rid of.

As far as I know marine diatoms tend to have more fragile scales. So they would be the perfect test material for mild cleaning methods.

[Hobbyst46]

I once had assembled a collection of smelly diatom samples that awaited cleaning . To speed up the process I incinerated them directly on a big stainless steel plate of about 12x12cm in my oven. The samples had quite some volume and I had to scrape the sample of the plate with a sharp knife. Together with the sample I removed quite a bit of dark grey scale from the stainless steel itself.

This is what I imagined upon reading yours and Fan's comments here, minerals and ashes can stick to metal surfaces.

So I would not advise to put the sample directly onto a piece of steel. A ceramic tile might be interesting as long as it is robust enough. What is used in a laboratory to heat samples up to 450°C? Special glass ware or porcelain?

When using an oven, a ceramic tile would be fine. In the lab, the appropriate vessel for such operations is called a crucible and is made of porcelain, glazed ceramics, or (better) alumina, or platinum . A porcelain crucible comes with an appropriate lid. Look for Fisherbrand porcelain crucibles, they are heat resistant to over 1000 degrees C. In addition, you can heat the crucible over a gas flame if you like, but better have an interface in between, like a steel mesh or wire-porcelain triangle to absorb some of the heat. For me, oven or hot plate is not an option, I can only use a kitchen gas stove.
As I posted in the other - parallel now - thread, I prefer to try a flat large coverslip. And rinse the residue (hopefully powder) or brush it into a centrifuge test tube, which is convenient for treatment-rinse-treatment etc (even without actual centrifugation, which I now avoid).

When using a deeper vessel, it could be possible to use it throughout the cleaning steps

Yes, the crucible is of sufficient chemical resistance for the relevant chemicals, BUT: It is not transparent, so the status of the diatom precipitate/flocculate is not easily visible, in contrast to a transparent test tube. Also, the dimensions of the crucible is not convenient for separation of solids and decantation. I would stay with test tubes.

I think that chemical cleaning should be easier after incineration than before. I would expect ashes to be powdery and alcalic, easy to get rid of.

Hopefully so.

As far as I know marine diatoms tend to have more fragile scales. So they would be the perfect test material for mild cleaning methods.

Indeed, I applied the mild cleaning to marine diatoms.
Now working on phase contrast photos of them and hope to post soon.

[zzffnn]

I once had assembled a collection of smelly diatom samples that awaited cleaning . To speed up the process I incinerated them directly on a big stainless steel plate of about 12x12cm in my oven. The samples had quite some volume and I had to scrape the sample of the plate with a sharp knife. Together with the sample I removed quite a bit of dark grey scale from the stainless steel itself. .......

I had the same experience, so I bought a ceramic coated cooking pan (Greenpan Thermolon, from grocery store Target in Houston/ Texas / US). It is supposed to be resistant to up to 450C: https://www.greenpan.us/why-ceramic

There should be other high temperature cooking pans with similar heat resistance.

We can always scrape with a hard plastic utensil, when everything cools down.

[Hobbyst46]

I once had assembled a collection of smelly diatom samples that awaited cleaning . To speed up the process I incinerated them directly on a big stainless steel plate of about 12x12cm in my oven. The samples had quite some volume and I had to scrape the sample of the plate with a sharp knife. Together with the sample I removed quite a bit of dark grey scale from the stainless steel itself. .......

I had the same experience, so I bought a ceramic coated cooking pan (Greenpan Thermolon, from grocery store Target in Houston/ Texas / US). It is supposed to be resistant to up to 450C: https://www.greenpan.us/why-ceramic

There should be other high temperature cooking pans with similar heat resistance.

We can always scrape with a hard plastic utensil, when everything cools down.

To bring the cooking pan to 450, you may need the highest stove flame, and even then, I fear that it will not reach much over 250 anyway. Pans are built to conduct heat and spread it. It might be heat resistant, for a safety margin, but frying in oil generally means about 250C, the boiling point of oil. Note that frying at around 250 creates those sticky black asphalts and charcoal residues in the pan. The smaller the incineration vessel, the higher the temperature, and the easier it will be to brush or rinse the diatom residue into a container for post-processing.

[zzffnn]

^ You may be right, though I have not test that or measured temperature.

[Hobbyst46]

Further notes regarding incineration:

1. When using the protocol of incineration AFTER chemical cleaning, and the diatoms are on coverslips, I advise to avoid thin metal heating plates. How thin? A 2mm thick stainless steel plate worked fine. Under the same conditions, I tried a 1.7mm thick plate and it slightly deformed within 10 minutes and lost its flatness. If it is not flat, heating the glass slips will be non uniform.

2. When heating diatoms, at the temperature that diatom frustules already soften and deform, the burnt residue is expected to be a mixture of minerals and charcoal (hopefully not black tar/asphalt). The black charcoal does not dissolve in HCl. It is attacked by 30% H2O2, but slowly. Hence, if trying the protocol of incineration BEFORE chemical cleaning, I would try to get rid of the black stuff prior to further cleaning with chemicals.
On my to-do list!

[Hobbyst46]

To pour some more fuel onto the diatom camp fire, here is a link to a quite recent research paper from Belgium where they incinerated as well as chemically cleaned benthic marine diatoms.
https://www.researchgate.net/publicatio ... phic_areas

They used a muffle furnace to reach 500 degC for 30 min. Their aim was identification of genera and species. Unfortunately, there are no images in the paper, although it was done with light microscopy. Their conclusions make me wonder about the true temperature limit for incineration. All the more so, since they incinerated very delicate and sensitive diatoms from the oligotrophic Mediterranean Sea, whereas I incinerated freshwater diatoms from a fish pond...where food should prompt a nice buildup of the silica skeleton (wild guess).

[zzffnn]

2. When heating diatoms, at the temperature that diatom frustules already soften and deform, the burnt residue is expected to be a mixture of minerals and charcoal (hopefully not black tar/asphalt). The black charcoal does not dissolve in HCl. It is attacked by 30% H2O2, but slowly. Hence, if trying the protocol of incineration BEFORE chemical cleaning, I would try to get rid of the black stuff prior to further cleaning with chemicals.
On my to-do list!

Maybe that black charcoal is produced by uneven temperature gradient across steel plate? I had that too, but I probably did not reach 350C when I tried it. That black charcoal may only be removed by very strong oxidation (difficult to do it safely)?

Was there much less black charcoal, when diatom sample was chemically cleaned first?

Thank you!

[Hobbyst46]

Maybe that black charcoal is produced by uneven temperature gradient across steel plate?

Possibly.

That black charcoal may only be removed by very strong oxidation

I guess so; either much higher temperatures (which may in turn damage the frustules or not), or hot 98% H2SO4 or 70% HNO3, each of which converts C to CO2. H2O2 also reacts with charcoal, but very slowly and the products are mainly solid organic molecules, not CO2. Permanganate or dichromate or hypochlorite will not work at all.

Let me re-phrase my observations and guesses for future work.

My observations:

a. All was done on the tiny amount of raw live stuff that remains on the 18x18 coverslip after drying, and looks like the photos above, that is - a really small amount of diatoms per incineration.

b. Incinerated diatoms themselves appear to be clean, some of them (needle-like forms) are bent, so they softened.

c. Incinerated samples, whether pre-cleaned (chemically) or not, contain non-diatom debris, most of it darker in color than diatoms (see more photos of the slide, below, where debris is marked with red circles). Under oblique illumination, some small dots appear to be black.

Was there much less black charcoal, when diatom sample was chemically cleaned first?

I think so, since relative to the amount of frustules, the debris seemed less for cleaned vs uncleaned samples; but this is not proven. And the difference was not large.

d. Chemical cleaning (EDTA+SDS) did extract brown/orange/green/gray pigments out of the initial collected sample. So the remaining residue must consist of less organic matter than samples that were not chemically cleaned. The extracted pigments definitely came out of the diatoms, and maybe also from non-diatom "dirt".

Thoughts about incineration followed by cleaning:

1. I do not see how to rinse an incinerated small coverslip, bearing a tiny amount of diatoms in the appropriate density for viewing, with a cleaning chemical
liquid without washing all stuff away from the glass and losing it.

2. If instead, a relatively large mass of an initial sample of collected diatoms (separated from water by sedimentation) is placed on a large (60x24mm?)
coverslip and incinerated, it may be possible to scrape that dry residue or brush it off into a small centrifuge test-tube (that far I can try to do, once I get
the plate and collect fresh plant diatoms). Then, add a few drops of 98% H2SO4, and dip the tube in a small warm water bath. I believe it will remove all
charcoal and most other detritus as well.

3. Do the same with 30% H2O2 instead of H2SO4 - very low chance, perhaps with longer heating time. Will not remove charcoal.
4. Do the same with EDTA+SDS - Will not remove charcoal. In addition, foaming may be troublesome.

(difficult to do it safely)?

Storage and use of strong oxidizing acids is hazardous, as we know; if one manages to store and use just a few drops of acid, taking the right precautions...not for me anyway.

The photos are of the same incinerated sample - no prior cleaning of any sort. 40x. The "oblique" is just a condenser position that increased contrast.

[Hobbyst46]

And here are some more photos of incinerated (only) diatoms on the slide. They are too dense but the image supports IMO the main previous conclusions:
1. most diatoms are clean
2. some diatoms (the long needle like, but also some of the small boat-like) are deformed (had softened)
3. there is some dark colored non-diatom debris - even a black one around 8 o'clock in set 1. I do not know what the yellow, orange and red spots are; perhaps incompletely burnt - caramelized - residues? iron oxides or other minerals plus silt?.
4. diatom chains are preserved

The photo is a stack of 3 images. Objective: 63x1.25 Neofluar (NO iris) oil immersion. Edge Oblique Light.

[Hobbyst46]

To pour some more fuel onto the diatom camp fire, here is a link to a quite recent research paper from Belgium where they incinerated as well as chemically cleaned benthic marine diatoms.
https://www.researchgate.net/publicatio ... phic_areas

They used a muffle furnace to reach 500 degC for 30 min, and warned against temperatures higher than 538C....
Their conclusions make me wonder about the true temperature limit for incineration. All the more so, since they incinerated very delicate and sensitive diatoms from the oligotrophic Mediterranean Sea, whereas I incinerated freshwater diatoms from a fish pond, where minerals should prompt a nice buildup of the silica skeleton (wild guess).[/quote]

Because of the results in that article, I wanted to double-check my thermometer. Previously, I had verified that it showed 0 and 99-100C, when the sensor tip was dipped in ice water and boiling water (at sea level), respectively. Today I verified that it shows correctly at 420C as well. So, IMHO, my measurements of incineration temperature were pretty accurate. Since that very incineration resulted in some bent and twisted frustules, I believe that 500C does soften the frustules of at least some diatoms.

I also repeated my heating experiment, using a new 70mm x 50mm (3500 sq-mm area) stainless steel plate, of thickness 3.0mm. A piece of scrap metal, from a different source. I placed it on the same wire mesh, over the maximum flame of the same stove burner I used before (my home stove). The results quite resembled the previous ones: the plate reached 440-460C within 7-8 minutes, then leveled off. The plate stayed flat and was not deformed.

I repeated this experiment, using the minimum flame of the same stove burner. The plate reached a roughly stable temperature of 350-400C within 8 minutes, and leveled off at 380-420C for at least 6-8 minutes afterwords.

So, for heating diatom-bearing coverslips, at temperatures of about 350-400C, I suggest a 2mm-3mm (not less than 2.0mm) thick stainless steel plate of 4000-4500 sq-mm area, if the stove flame is similar to mine (home propane/butane fuel gas, flame diameter 7cm).

Verification of the plate temperature requires an appropriate thermometer. Alternatively, place a tiny bead (3-4mm size) of a melting point standard on the plate, near the incinerated coverslips.
MP standards:
Pure Zinc pellets (sold by chemical suppliers) melt at 419C.
Pure Lead (hold the pellets with tweezers, not fingers!!) melts at 327C.
Less accurate standards, but easier to buy - soldering alloys or wires, that are graded according to their melting points.

Under heating, the bead starts to melt when the plate has reached the temperature. If in doubt, touch the bead lightly with a thin long screwdriver blade to verify. None of the above metals combines with stainless steel, so they can be scraped and removed after cooling.

Caution: The plate remains extremely hot for a long time. It can be rapidly cooled by securely holding it with isolated pliers and rinsing (far from the face!) under running tap water.

[zzffnn]

I did a quick and dirty home experiment, with an oven thermometer with highest possible reading at 350C, a ceramic coated cooking pan rated at 500C and my kitchen (natural gas) stove fire. Thermometer sat directly on and touching cooking pan surface.

In about 12-15 minutes, the thermometer read 325C and seemed to stop there. I stopped because I saw some black fluid coming from edge of my cooking pan. Maybe that was just cooking oil dripping from my ventilator above.

So I am guessing the pan surface can get to about 325-350C, assuming my cheap thermometer is not off too much.

Whether or not that temperature is good enough for diatom cleaning, I am not sure. I did see some black charcoal remained.

[Hobbyst46]

Hi zzffnn - glad to find you online!
natural gas and condensed hydrocarbon gas flames yield about the same calories if I trust Wikipedia.
the pan has really outdone itself, given its large area - unless you are using the strongest flame of the stove?
if the pan has been unused before, perhaps the handle (bakellite?) was overheated and melted?
I am pleased so far with my scrap metal stainless steel plate - it is reproducible and is heated above 400C
My cooking pans are all Teflon coated. Teflon decomposes at 250-300C and yields toxic vapors. So I cannot repeat your test...sorry.
Hope to soon collect more diatoms from Nature and incinerate them in quantity as mentioned.

[zzffnn]

Yes, I did use highest fire height I have.

The oven thermometer was sitting unstably on the cooling pan. So maybe pan surface got to slightly higher temperature, around 350C, instead 325C?

No, the pan handle did not melt and it has been used before. I moved the handle away from flame and have been watching it. I think it was just drip from my dirty kitchen ventilator (that I was cleaning at the same time).

There are uncoated (camping) steel cooking pans that look thick enough. Maybe someone can get the smallest possible size and give it a try?

[MicroBob]

When I incinerated bigger samples I had the impression that incineration didn't work as well as with thinly spread samples. May be the oxygen can't reach the lower layers. I was thinking about improving oxydisation by supplying pure oxygen to the sample. I have no idea how to calculate the needed amout of oxygen though. Is one of you able to calculate how much oxygen is needed for 1g of organic material? I'm on holiday now and brought some containers and a tiny Swift field microscope. I will for sure bring some diatom material back home.

[Hobbyst46]

When I incinerated bigger samples I had the impression that incineration didn't work as well as with thinly spread samples. May be the oxygen can't reach the lower layers. I was thinking about improving oxydisation by supplying pure oxygen to the sample. I have no idea how to calculate the needed amout of oxygen though. Is one of you able to calculate how much oxygen is needed for 1g of organic material?

Hi Bob,
I do not know the exact answer to the question that I bolded, but a crude super-simplistic theoretical calculation can be done:

From a web source I fished out that alga consists of 56% carbon, 6% hydrogen, 8% nitrogen and 31% oxygen.
I assume that your sample is 1g of dry alga.
I convert C to CO2, N to NO2, H to H2O, calculate the total amout of oxygen for the transormation, and subtract the small amount of oxygen that is already in the diatoms, and the result is 1.8 liter of oxygen at ambient conditions.

Practically, though, I think that the underlined text is indeed the reason, and IMHO the first way to fight it is prolonging the incineration.

[Hobbyst46]

Yes, I did use highest fire height I have.

That explains the very high temperature you get.

The oven thermometer was sitting unstably on the cooling pan. So maybe pan surface got to slightly higher temperature, around 350C, instead 325C?

could you post a photo of the thermometer and its sensor? anyway, a temperature gradient of 20-30C along the pan is definitely likely, perhaps even more. How long did it take to get that temperature?

There are uncoated (camping) steel cooking pans that look thick enough. Maybe someone can get the smallest possible size and give it a try?

I can search in camping stores; ordinarily I see mostly cast iron pans or coated aluminum pans.