Doing Diatoms

[zzffnn]

Right members I think I have the answer (I could be wrong) but I would put my shirt on it being Biddulphia this was obtained from the 'diatoms of the inland waters of temperate North America' it also states Solitary or in straight or zigzag chains. Mostly Marine and Rare.
That's all folks.

http://westerndiatoms.colorado.edu/taxa ... ira_laevis

It is the same diatom mentioned by Rod:

Pleurosira laevis
(Ehrenberg) Compère 1982 Category: Centric
BASIONYM: Biddulphia laevis Ehrenberg 1843
SYNONYM(S): Cerataulus laevis (Ehrenberg) Ralfs in Pritchard 1861

[rnabholz]

Right members I think I have the answer (I could be wrong) but I would put my shirt on it being Biddulphia this was obtained from the 'diatoms of the inland waters of temperate North America' it also states Solitary or in straight or zigzag chains. Mostly Marine and Rare.
That's all folks.

http://westerndiatoms.colorado.edu/taxa ... ira_laevis

It is the same diatom mentioned by Rod:

Pleurosira laevis
(Ehrenberg) Compère 1982 Category: Centric
BASIONYM: Biddulphia laevis Ehrenberg 1843
SYNONYM(S): Cerataulus laevis (Ehrenberg) Ralfs in Pritchard 1861

A rose by any other name......

Well it would seem we are in agreement. Thanks exmarine and zz.

[rnabholz]

Rod,

I like your brightfield stack.

I improved my live mount techniques today and carefully screened my latest samples collected this Wednesday 21st (which came from more shallow water and different collection sites, compared to what I sent you). I did not see many interesting forms (after making 8 mounts of different precipitation depth), so not sending them this time.

The key to live mount improvement, in my case, is using less raw material, in more diluted and teased apart manner, making thinner mount and viewing in oblique light. That worked very well, even without cover slip (at NA 0.20 - 0.40). Previously, I viewed in darkfield, used thick and undiluted samples, which caused too much optical obstruction, halos and flares. Phase probably won't work well in that case either.

Thanks zz

I experienced the same realization, that less is generally better and teasing is the indispensable step to create the best and most views on each wet mount.

[zzffnn]

Nicely done Fan.

Boy, I'll say!

So #1 & #3 look as though single frames were unable to gather it all in, due to extraordinary depth? Or is something else going in? I'd like to zero in on those ocelli features. I definitely want a few slides of that! I can see where it may also become something of a case study, where either Fan or I shall gather samples periodically to study annual cycles, something I've always wanted to do but never quite got around to.

TAMU did a diatom annual cycle study in Galveston (Texas) here:

http://www.tamug.edu/phytoplankton/Rese ... n_Bay.html

Here is an abstract of what they found:

we did find that specific genera of diatoms and dinoflagellates, the two major groups, occur in specific parts of Galveston Bay at specific times of the year. For example, the diatoms Navicula, Thalassionema, Thalassiosira and to a lesser extent Pleurosigma, were most abundant at Stations 1 and 4 (Bolivar Pass near mouth and Morgan’s Point in the middle of Galveston Bay respectively – see map below) during the spring and summer months (March to August 2005).

But do search with "diatom" as keyword on that web page and you will find more information.

[exmarine]

Wish I could join in with the debate but I am in hospital having my fourth knee joint replacement done in Cardiff, Wales, and I live in Cambridge, England. The reason for this is because the top surgeon for sorting out other surgeons cock-ups is based in Wales.
Enough of my woes members keep the diatoms rolling they cheer me up.
Thanks guys.

[rnabholz]

Wish I could join in with the debate but I am in hospital having my fourth knee joint replacement done in Cardiff, Wales, and I live in Cambridge, England. The reason for this is because the top surgeon for sorting out other surgeons cock-ups is based in Wales.
Enough of my woes members keep the diatoms rolling they cheer me up.
Thanks guys.

Sorry to hear about the troubles. Best wishes for a speedy and complete recovery.

Hurry back.

Rod

[zzffnn]

I found lots of diatoms yesterday, from a branch of Cabomba/fanwort plant in a sun-lit area of Herman Park's pond in Houston (Texas, USA). High temperature is in the upper 80s for October in Houston. But there is almost no diversity (almost all are long/slender kayak shaped).

I would probably use those diatoms to practise cleaning (by burning them to ash in a steel container).

Edit:

So I cooked them to ashes. But that only cleaned them partially. Diatoms are now in skeleton form (without chlorophyll), which made them more difficult to see. Other organic stuffs were reduced significantly. But the problem is lots of non-diatom stuffs still remain. I guess a propane hand torch is needed for better cleaning.

Cooking to ashes + oxidative reagents should also work, theoretically more efficiently than oxidation/boiling alone.

I found a few more types (that we have seen before), other than the long/slender kayak form, after scanning burned sample with a 20x inverted objective.

[rnabholz]

I found lots of diatoms yesterday, from a branch of Cabomba/fanwort plant in a sun-lit area of Herman Park's pond in Houston (Texas, USA). High temperature is in the upper 80s for October in Houston. But there is almost no diversity (almost all are long/slender kayak shaped).

I would probably use those diatoms to practise cleaning (by burning them to ash in a steel container).

Edit:

So I cooked them to ashes. But that only cleaned them partially. Diatoms are now in skeleton form (without chlorophyll), which made them more difficult to see. Other organic stuffs were reduced significantly. But the problem is lots of non-diatom stuffs still remain. I guess a propane hand torch is needed for better cleaning.

Cooking to ashes + oxidative reagents should also work, theoretically more efficiently than oxidation/boiling alone.

I found a few more types (that we have seen before), other than the long/slender kayak form, after scanning burned sample with a 20x inverted objective.

I am interested to hear about your experiences with incineration. Kurt has threatened to give it a try, but I don't think he has pursued it yet.

[zzffnn]

Rod,

I don't think I am buying a propane torch just for diatom cleaning. We don't currently have space for that much gears.

So I won't try incineration, unless I find a cheap/easy/safe way to burn at higher efficiency/temperature. The temperature I got was not enough (to effectively remove other organic debris by itself), with my kitchen stove.

Kurt might have a hand torch.

But, I think even my way of heating/burning can make your current cleaning steps shorter and more efficient. Just add burning (to ashes) before your chemical treatment steps. That should reduce organic debris very significantly. I saw it that way, with naked eyes and under scope.

[rnabholz]

This evening I got to the sample from Langdon Beach, Pensacola, Florida that was sent to me by zzffnn.

It was a ziplock sandwich bag filled about a third of the way with damp sand with the notation:

August 2016
Langdon Beach
Pensacola FL
Submerged sand
Surf front
Afternoon

It it was some very fine whitish sand with very small pieces of shell mixed in.

I placed about three tablespoons of the sand in a jar, and added water until I had about an equal level of water over the sand. I then gently swirled the mixture for about a minute. Upon stopping, I held the jar up to the light and I could see that the water was clouded. After about 20 seconds, after the sand had settled, I very slowly and gently poured off the water, and repeated the process again with the same sample. The second effort was rewarded with a much less cloudy water product which was also poured off.

I continued that process with the remaining sand in the sample, yielding this beaker of what I hope will be diatom filled water.

Diatoms in Suspension

Langdon Beach Suspension.JPG (139.82 KiB) Viewed 193573 times

After letting the sample in the collection container settle for about 20 minutes, I could see material beginning to settle on the bottom, but not a great deal off it. I used a pipette to gather a bit for a wet slide to see just what I had.

There are diatoms in the mix. A good variety, with some centric colonials visible as well other interesting forms that were unmistakable marine forms.

I will let the sample settle overnight and see just what I end up with. My concern at this moment is that it will not produce material of sufficient volume that I will be able to clean it without losing the greatest portion in the process of cleaning and rinsing. We'll see what things look like tomorrow.

[zzffnn]

If you feel there are not much other organic stuff, you may not need to clean the sample that much?

I am guessing 3 tablespoon of sand will probably get you around 3-6 sparsely mounted slides, based on what I saw.

Also, since those diatoms are small, light and not in high quantity, centrifuging them to wash them may be safer than pouring out wash water.

Please see my monkey darkfield images in this thread:
viewtopic.php?f=6&t=3760&p=34169&hilit=Pensacola#p34169

That (very sparse) density was produced by half of a tablespoon of sand. That quantity is good enough for me though.

[rnabholz]

If you feel there are not much other organic stuff, you may not need to clean the sample that much?

I am guessing 3 tablespoon of sand will probably get you around 3-6 sparsely mounted slides, based on what I saw.

Also, since those diatoms are small, light and not in high quantity, centrifuging them to wash them may be safer than pouring out wash water.

Please see my monkey darkfield images in this thread:
viewtopic.php?f=6&t=3760&p=34169&hilit=Pensacola#p34169

That (very sparse) density was produced by half of a tablespoon of sand. That quantity is good enough for me though.

They will need cleaning to release the girdle bands to separate the valves, clean out the valves and break up the colonials. Many of the diatoms I saw were trapped in clumps of organic material, so those should benefit as well.

I will of course centrifuge for the rinse, I need to keep the good stuff down while taking off the supernatant.

I will do my best, but there is not much room for error with this batch.

[zzffnn]

No pressure and no rush at all, Rod. You don't have to produce anything out of that sand sample of limited quantity. There was not much sand to start with - I had only double the amount of what I gave you, then I gave half each to Kurt and you.

At worst, it would give you a practice run before Dale's Maui sand gets to you.

[rnabholz]

Returning to the bench tonight, the sample has settled nicely.

I thought some today about the best way to take off the extra water to reduce the volume to a size that I could centrifuge.

Pouring it off was out of the question, I could not spare any of the diatoms that might escape. Using the disposable plastic pipettes could also be problematic as a small slip of the hand that caused an unintended outward squirt could resuspend the diatoms I had so patiently waited to settle.

LB Pipette.JPG (82.19 KiB) Viewed 193531 times

I decided to use my pipette bulb. The bulb makes it very easy to control the direction of the flow and makes in very difficult to make a mistake. The downside is that it is very slow when you have to remove that much water. As slow as it was, I was able to draw water precisely from the top of the column avoiding drawing in the settled matter on the bottom.

I took the water down to about 30ml, which showed a remaining sample with some promise.

LB Product.JPG (67.06 KiB) Viewed 193531 times

From there I transferred the material to two 15ml centrifuge tubes

LB Tubes.JPG (101.26 KiB) Viewed 193531 times

Then placed the opposite of each other in the centrifuge

LB Centrifuge.JPG (84.52 KiB) Viewed 193531 times

Then spun them for about 10 minutes on a relatively low setting around 300 to 400 rpm. When I shut it down and took out the tubes, I was pleased to see this.

LB Spun.JPG (68.41 KiB) Viewed 193531 times

Much of this brown material is organic material, but I know from my look at the live sample that it contains a pretty nice bunch of diatoms too.

[rnabholz]

Transferred the sample to a small beaker and it was ready for Hydrogen Peroxide

LB HP.JPG (97.15 KiB) Viewed 193531 times

I added an equal volume of HP to the sample. There was no immediate reaction, most likely because this sample is pretty clean of organics and minerals. I will likely let it sit overnight and then add the Potassium Dicromate tomorrow.

Looking good so far....

LB In HP.JPG (80.03 KiB) Viewed 193531 times

[zzffnn]

Rod,

You don't have enough centrifuge tube, I assume? I think I have some extra 15 ml plastic centrifuge tubes.

Since you have 6 slots there in your centrifuge, why not reduce volume to 15x6=90 ml, spin with 6 tubes, instead of spinning only two tubes of 30 ml in total?

You can spin at higher speed, say 1000 rpm, as there are no super delicate forms. A more compact pellet will allow you to remove even more water and save oxidation reagents.

Just my 2 cents. What you did was perfectly fine and would work the same way, likely better, just a little slower.

[rnabholz]

Rod,

You don't have enough centrifuge tube, I assume? I think I have some extra 15 ml plastic centrifuge tubes.

Since you have 6 slots there in your centrifuge, why not reduce volume to 15x6=90 ml, spin with 6 tubes, instead of spinning only two tubes of 30 ml in total?

You can spin at higher speed, say 1000 rpm, as there are no super delicate forms. A more compact pellet will allow you to remove even more water and save oxidation reagents.

Just my 2 cents. What you did was perfectly fine and would work the same way, likely better, just a little slower.

I have plenty of tubes.

My rationale was that I could safely reduce the sample to 30ml without losing any diatoms, and the fewer transfers between containers, the less likely I was to lose any by spill or being left behind in a container.

As to the spin speed, I could get the result I needed at a lower speed, the pellet will need to be separated for cleaning anyway and the total volume was 60ml before the spin, after decanting the supernatant and combining the contents of both tubes it was less than 20ml. It won't take much by way of chemicals to clean that small amount.

[rnabholz]

This morning I added the Potassium Dichromate to the mix and left it to cook while I was at work.

I returned this evening to find that nice orange mix that indicated that the reaction was completed. A quick wet slide confirmed that the sample was clean. From there it was just five rinse cycles to produce the finished sample.

I made a couple of permanent slides and the resulting strews are quite nice. Nice mix of forms, various sizes and shapes. Here are a few hastily located and shot images, as I was running out of time for the evening.

AO 20 100x Phase Plan Achro Oil, Phase Condenser, Canon 70D Single Frames

Sorry no ID's - short on time and still need to find a good Marine reference.

63µm

Pensacola 1993.JPG (194.88 KiB) Viewed 193493 times

66µm

Pensacola 1999.JPG (88.04 KiB) Viewed 193493 times

29µm

Pensacola 1996.JPG (115.84 KiB) Viewed 193493 times

46µm

Pensaocola 1997.JPG (89.05 KiB) Viewed 193493 times

[zzffnn]

Great job, Rod. Very nice images too!

[zzffnn]

Rod,

Did you see any small foraminifera?

Please take a look at my images:

viewtopic.php?f=6&t=3760&p=34169&hilit=Pensacola#p34169

In my "1.jpg", there is one at around 3:30 o'clock position;

2.jpg, there are 3 at around 5 o'clock, 6 o'clock and 9 o'clock positions;

4.jpg, there is one at around 5 o'clock position;

6.jpg, there is one close to frame center.

[rnabholz]

Thanks zz

I did not have much time to really look closely tonight, just a cursory scan and quick images. I did not see any forams, but they could have been there. I will have a better chance tomorrow.

[zzffnn]

No worries and no rush, Rod. I was just curious if your diatom cleaning/mounting procedure would work the same for small forams.

[rnabholz]

I did find this scanning the Pensacola sand with the stereo scope. I believe it was about 60x.

Please excuse the phone resize and edit.

[rnabholz]

More images from the Pensacola sample here:

viewtopic.php?f=6&t=3956&p=36122#p36122

[rnabholz]

Another trip to Wisconsin over the weekend and another chance to gather a few samples.

The Mecan River is a small meandering stream, clean and clear. Finding an obvious source of diatom material was a bit of a challenge. Could not find that characteristic brown/black coating on rocks and sticks, at least not at first glance. I pulled some submerged vegetation out of the water, and although it appeared clean, rubbing it between my fingers I could feel the slight grittiness that I had experienced in handling diatom rich water. So I grabbed a good size bundle of that plant matter and tossed it in the bucket.

The nearby Upper Fox River is a larger stream with a more turbid water. The material I found there was much easier to spot, as the recognizable coating was prevalent in may places. Again I chose vegetation rather than rocks to keep the mineral content down.

Finally, Green Lake, Located in the City of Green Lake, County of Green Lake....a bit redundant huh? A 7900 acre lake, and the second deepest in Wisconsin at 236 feet. I found a small protected niche that had a thick coating of diatom material covering everything that was easily visible through the wonderfully clear water. I snagged more vegetation from the bottom that was about 4 feet down.

Returning home, I separated the diatom material from the vegetation using the shake method, place the vegetation and some water in a jar, seal, and shake until the water in the jar shows cloudiness. Drain of and save the water and discard the vegetation.

All of the samples produced good diatom material with the Mecan sample being the least dense, but that was as expected based on the nature of the sample.

Under the scope, the wet slides all showed good diatoms. Here is a quickie afocal 20x snapshot of the Fox River sample.

Fox River Sample 20x.JPG (233.8 KiB) Viewed 193411 times

All of the samples will be getting the cleaning treatment. More to come as the process continues.

One other development. As the weather cools and some of the water bodies have receding water levels, gathering samples had become a bit harder to do without getting wet or muddy feet. I devised a tool the extend my reach and used it over the weekend to gather the samples and it worked out really well.

The main component is an extendable pole designed to assist in changing light bulbs that are difficult to reach. It had been living in the garage for some time just waiting for a bulb to burn out. It is something like this:

https://www.amazon.com/Ettore-48450-Bul ... +bulb+pole

Mine is about 12 feet long fully extended. To that I added one of these:

https://www.amazon.com/Bond-1501-3-Tine ... cultivator

I got the cheap one at the local farm store, about $3 or so.

With a generous application of some good duct tape, the claw was attached to the end of the pole. I now have a reach of somewhere in the 14 foot range (counting my arms). At the lake, I was easily able to reach down 3 to 4 feet in the water and gather material from the bottom. On the Mecan, I stood on a low bridge over the stream and reached down about 8 feet and snagged my sample, and the on the Fox I was able to reach out around 10 feet to get the goods. All while staying high and dry.

The claw can also easily drag baseball size rocks to shore should that be necessary.

I am really pleased with it, thought some of you might be interested.

[zzffnn]

Thank you for sharing, Rod.

That claw is a good idea, especially for vegetation and small rocks.

I wonder what is the best tool/method for scraping submerged big rocks (>25 lbs). Imagine wading and weightlifting at an oyster reef. That was kind of what happened when I collected my Galveston Offatts Bayou sample. Rocks there were not quite as sharp as oyster shells, but not far behind - I ended up with multiple cuts on my legs and hands (I forgot to take gloves and rain boots).

If you only collect diatoms from vegetation, you may be missing diatoms that prefer to live with other rock algae. I read that different diatoms have different preferred host plant/site. Collecting from different host plants may help too, if you care about diversity.

I joined the Yahoo diatom site and was referred to this fossil diatomite sites list: https://groups.yahoo.com/neo/groups/dia ... Diatomite/

I wonder why they don't have a list for recent sample sites? Is it because you can find recent diatoms everywhere? I am guessing a good modern site would not offer less diversity, concentration and integrity (integrity could he better).

[rnabholz]

Easy Fan, no need to risk injury for this cause!

In places with a current I have seen the use of a net to catch material liberated from a rock by scraping. Not sure how tight a mesh you would need for diatoms and whether you could get water to flow through like you would need it to do.

You are correct about collecting from varied surfaces for maximum diversity. While I did collect primarily vegetation, I did collect both live plants and dead material from the bottom. Hopefully that earned me some of the benthic varieties.

I would guess that the issue with listing live collection sites would be trying to decide what puddle of water you might exclude, as they all have diatoms to some degree more or less.

The Clawpole (Poleclaw?) Really worked well for me. It made access to the material very easy. One aspect that I didn't mention, but you actually touched on is safety. As hard as it may be to believe, some people don't find collecting diatom samples very exciting (Whaaaaaaat?), So I am often out alone, and in some out of the way places. The prospect of a fall on slippery mud, or a turned ankle negotiating rocky shorelines is very real. Anything that helps avoid that possibility is a good thing.

Thanks for the interest.

Rod

[exmarine]

Hi All you guys over the pond,

You have been busy and with good results as shown by the photos, also thanks for sharing the methodology for collection.

Really interesting reading the posts and studying the diatoms to come to a conclusion as to the generic make-up.

Thanks guys.

Exmarine.

[rnabholz]

Hey exmarine,

Good to see you back in action.

My season is soon coming to an end, but maybe I can count on my southern compatriots to keep me busy over the winter.

Glad you are enjoying the posts, wasn't sure anyone but zz and I were still around. ;^)

Rod

[rnabholz]

I was able to make a mount of the Green Lake sample tonight. Overall a nice variety of diatoms, no really large ones but lots of interesting midsize and smalls. The sample does contain more minerals that I would have preferred, but they are manageable.

Here are a few shots from the very first slide.

Centric 45µm

Green Lake Centric 2241.JPG (141.94 KiB) Viewed 193344 times

Nitzschia 90µm

Green Lake Nitzschia 2240.JPG (104.62 KiB) Viewed 193344 times

Caloneis Amphisbaena 63µm

Green Lake Caloneis Amphisbaena 2248.JPG (96.83 KiB) Viewed 193344 times

Cymatopleura 66µm

Green Lake Cymatopleura 2245.JPG (83.65 KiB) Viewed 193344 times

Nitzschia 80µm

Green Lake Nitzschia 2244.JPG (82.92 KiB) Viewed 193344 times

More images from the Green Lake sample can be seen here:

viewtopic.php?f=6&t=4072

viewtopic.php?f=6&t=4085