Doing Diatoms a Different Way

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KurtM
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Re: Doing Diatoms a Different Way

#91 Post by KurtM » Wed Jan 25, 2017 4:22 pm

Charles, thanks for posting this very interesting experiment!

You say added 1 ml diatoms to 15 ml water - do you mean a thick concentration of diatoms measuring 1 ml in volume, or 1 ml of pure diatoms? I imagine you are handling them as suspensions, with pipettes? I am always very interested in hearing details of how people do things in the lab, as I have extracted lots of great ideas and inspiration from such discussions.

I am also curious as to the cross section of the original sample found after each settling interval? I mean, heavier forms sink faster than lighter ones, and I suppose settling times might amount to a form of sorting?

In my continuing efforts to discover means of separating mineral content from diatoms, I experimented with swirling in Petri dishes, similar to "panning for gold." In one memorable instance, I started with a sample rich in beautiful large Cymbellas and swirled away, sucking up the diatoms around the perimeter and leaving the sand at the center. Under the microscope, however, I found all my Cymbellas had disappeared! I quickly relocated them: they had migrated to the center with the sand. I never was entirely sure how to interpret that little finding, except to reinforce my general aversion to separation efforts as resulting in loss of desirable material. Well, except for picking them out individually, of course.
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Re: Doing Diatoms a Different Way

#92 Post by rnabholz » Thu Jan 26, 2017 1:16 am

Hey Charles,

Thanks for sharing the results of your experiment. I was concerned after your previous posting that I was losing a lot of good stuff down the drain. It would seem now that things are pretty effectively settled after a relatively short time. Great to know.

As circumstances have it, I used my new syringe last night for the first time in a "live" exercise. I had raw material cleaned from the sample you sent from Buck Roe Beach in a 500ml beaker with about 400ml of water over the material that had settled overnight.

I was able to remove 300ml very quickly, and without disturbing the settled matter. I generally try to get the level down to 50ml or so. Removing the last 50ml, I drew the syringe plunger very slowly as the hose was just a matter of about 1/4 inch from the bottom.

After that process, the collected water was quite clear - hopefully that means that I left the good stuff in the processing beaker.

That experience made me think about the size of the container that I was using and how a smaller one might work better, but it is pretty common to have 400ml of water to process cleaning these sand samples. Ideally, when approaching that 50ml target volume, there would be more water above the material to make it less likely that any would be swept up by the syringe hose.

Then it occurred to me that a large graduated cylinder might be perfect for the job. The narrow diameter would stack as much water as possible above the sample, improving the "safety zone" for the hose end. I found and ordered a 500ml Graduated cylinder on ebay. Doing a bit of figuring it looks like there would be a bit over an inch of water in the cylinder when the level is reduced to 50ml, which should be plenty. We will see.

Thanks again for posting the experiment results- Very helpful!

Rod

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Re: Doing Diatoms a Different Way

#93 Post by KurtM » Thu Jan 26, 2017 1:29 am

Hey, I like the graduated cylinder idea, and the hose on the syringe makes it very possible to use. Practical, even! Awright, this is getting interesting... :geek: :geek: :geek:
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Re: Doing Diatoms a Different Way

#94 Post by Charles » Thu Jan 26, 2017 1:18 pm

Kurt, The diatom concentration was taken as approximate as it was the amount which settled into the tip of a 15 ml centrifuge tube. And yes, it seems like the heavier forms settled faster than the lighter forms. It is very hard and probably impossible to isolate all diatoms out of a sand sample as some diatoms are even heavier than the sand particles. For me, it is not a problem as I can pick the diatoms from the sand.

Rod, What a great idea on the graduated cylinder.

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Re: Doing Diatoms a Different Way

#95 Post by Charles » Thu Jan 26, 2017 1:49 pm

Looking through some lab glassware on ebay, I saw some separation flasks which look promising. These flasks come to a point at the bottom with a stopcock...hummmm?

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Re: Doing Diatoms a Different Way

#96 Post by rnabholz » Thu Jan 26, 2017 5:01 pm

This conversation has led me to consider a new protocol for cleaning and rinsing samples.

Charles' experiment seems to indicate that in a 15ml tube diatoms will settle in about 5 minutes. That was a surprise to me, as I expected that period to be much longer, on the order of hours.

Laboring under that belief, I was certain centrifugation was necessary to allow me to accomplish what I wanted to in the scarce time I had to devote to the process. Maybe not?

As I generally spun the samples for 5 to 10 minutes anyway, I could certainly spend the same time letting the sample settle naturally.

The other consideration was managing the wash fluids.

Using the small 15ml tubes meant that there was little to clear off for each round of cleaning, but of course that also meant that the volume of clean water that could be added each time was limited, and its cleaning capacity was therfore limited as well.

The use of the syringe changes that, making handling larger volumes of rinse water much quicker and efficient.

Soooooo, what I am thinking is this:

1. Use the 500ml Graduated Cylinder for the rinsing vessel.

2. Use the syringe to facilitate quick and safe draw off of the larger volume wash water.

3. Allow for natural settlement of diatoms - no centrifugation.

4. If a 5" 15 ml tube settles in 5 minutes, assuming a similar rate of fall, the 14" 500ml would likely settle in 15-20 minutes.

5. Filling the cylinder to 450ml would increase the rinse water capacity by approximately 30 times vs the 15ml tube, making for a much more effective dilution of the remnant cleaning chemicals - perhaps meaning that fewer cycles would be necessary - maybe leading to a net saving of time.

6. The diatoms in the sample would not be subjected to the force of centrifugation and the resultant required unpacking of the mass at the bottom of the tube for rinsing. That would very likely mean that we would see less breakage in the sample.

So what do you think? Are my assumptions reasonable? What am I missing?

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Re: Doing Diatoms a Different Way

#97 Post by billbillt » Thu Jan 26, 2017 6:48 pm

Hi Rod,

What you posted seems viable to me.. I was also surprised to hear that diatom precipitation took just a short interval of time.. Please post the results if you pursue these methods further...

BillT

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Re: Doing Diatoms a Different Way

#98 Post by rnabholz » Thu Jan 26, 2017 10:10 pm

billbillt wrote:Hi Rod,

What you posted seems viable to me.. I was also surprised to hear that diatom precipitation took just a short interval of time.. Please post the results if you pursue these methods further...

BillT
Thanks Bill,

It will be a few days until my graduated cylinder arrives, so progress will be halted until then. Standby...

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Re: Doing Diatoms a Different Way

#99 Post by zzffnn » Fri Jan 27, 2017 12:35 am

Thank you Charles for sharing your results.

Rod,
I doubt there would be any significant diatom damage, if you centrifuge at 100 round per minute (rpm). I used to centrifuge delicate human cells (with texture similar to Paramecium) at 400 rpm and they survived and thrived (did not break) just fine after that. But you surely have reason to be safe rather than sorry.
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Re: Doing Diatoms a Different Way

#100 Post by rnabholz » Fri Jan 27, 2017 1:14 am

Thanks zz.

I am afraid my centrifuge won't run that slow, so that is not an option.

In any event, I think the case is easy to make that natural precipitation would have to be easier them.

We will see how things work out.

Rod

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Re: Doing Diatoms a Different Way

#101 Post by Charles » Mon Jan 30, 2017 1:40 pm

A very bad week for me.

First, as I was separating out some forms on the species line, I inadvertently drug my needle through about six lines of forms, scattering a lot and even damaging a few.

Then trying to clean a very gunked up needle on a piece of lens paper, it got tangled in the strands and broke off.

Then replacing the needle, I didn't have it properly adjusted for height and proceeded to smash it into the slide breaking the new needle.

So, I took a break to go hunt for new specimens. I wanted some specimens from the South shore of the Chesapeake Bay and another river source which empties into the bay. I got a sample from a cove like area in Norfolk for my South shore sample and then took a sample from the Deep Creek River near my home. Both were at low tide, so I was able to get well out into the bay and river to collect my sand samples.

I processed the Norfolk sample not expecting it to be too different than the North shore Buckroe Beach sample but it was indeed very different. While the primary species from the North shore were Achnanthes, Actinocyclus, flat centrics like Coscinodiscus, Odontella with a few Auliscus and Rhaphoneis, the primary species of the South shore were mainly of several different species of Odontella/Biddulphia and lots of Auliscus.

Pictures to follow (give me a day or so Kurt! 8-) )

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Re: Doing Diatoms a Different Way

#102 Post by zzffnn » Mon Jan 30, 2017 2:31 pm

Sorry to hear about those accidents, Charles.

But that is an experience that only talented micro manipulators would share - ham-fisted people like myself won't even attempt such manipulation to begin with. I cannot even keep my diatom slides intact/clean and Kurt called me "wide man" for that :twisted:
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Re: Doing Diatoms a Different Way

#103 Post by rnabholz » Tue Jan 31, 2017 4:43 am

Oh my Charles, I can't imagine how you felt after scrambling one of your arranged slides! That must have been an awful moment.

I am with zz, that would likely be an hourly occurrence for me should I ever try picking. Strictly strews for me I think.

Look forward to seeing what you came up with in your new samples. Standing by....

Rod

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Re: Doing Diatoms a Different Way

#104 Post by Charles » Sun Feb 05, 2017 5:06 pm

Thank you Z and Rod. Accidents and mistakes happen. But on the bright side, I was able to rearrange and expand the type line which now has over 70 different diatoms.
Last edited by Charles on Sun Feb 05, 2017 5:23 pm, edited 1 time in total.

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Re: Doing Diatoms a Different Way

#105 Post by Charles » Sun Feb 05, 2017 5:22 pm

Some photos of the different forms in the Norfolk, Chesapeake Bay (South shore) forms:
Here is the storage slide with most of the forms at 2.5X setting on the SZ7:
R-Norfolk NS Storage1.jpg
R-Norfolk NS Storage1.jpg (80.31 KiB) Viewed 5775 times
The rest of these were taken at 7X setting.
The strews were loaded with Auliscus:
R-Auliscus.jpg
R-Auliscus.jpg (88.32 KiB) Viewed 5775 times
And loaded with these large menacing looking Biddulphia:
R-Odontella-Biddulphia.jpg
R-Odontella-Biddulphia.jpg (75.35 KiB) Viewed 5775 times
As well as these three 'eyed' Pleurosira:
R-Pleurosira.jpg
R-Pleurosira.jpg (62.71 KiB) Viewed 5775 times
There were very few other centrics:
R-Some Centrics.jpg
R-Some Centrics.jpg (61.61 KiB) Viewed 5775 times

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Re: Doing Diatoms a Different Way

#106 Post by Charles » Sun Feb 05, 2017 5:27 pm

There were some pennates as well:
R-Pennates.jpg
R-Pennates.jpg (65.42 KiB) Viewed 5772 times

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Re: Doing Diatoms a Different Way

#107 Post by rnabholz » Sun Feb 05, 2017 6:07 pm

Wow - what a variety you have collected!

The tri-sided types are something that I never see around here, and in fact have never come across. And the Biddulphia! They are huge.

Excellent work Charles. What a stock you have built. Time for you to get them under your compound and enjoy the intricate beauty of all of these wonderful forms.

Please keep reporting. Really enjoying reading about the approach you take.

Rod

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Re: Doing Diatoms a Different Way

#108 Post by KurtM » Sun Feb 05, 2017 6:15 pm

Yep, very inspirational alright! I swear one of these days I shall follow in these footsteps, if I ever get my butt back in my lab for any length of time. The weather has been entirely too pleasant around here.
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Re: Doing Diatoms a Different Way

#109 Post by Charles » Tue Feb 07, 2017 12:27 am

Thanks Rod and Kurt!

Yes, I do need to start putting these into permanent mounts. It will be happening soon.

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Re: Doing Diatoms a Different Way

#110 Post by Charles » Wed Mar 01, 2017 12:20 pm

I'm finishing processing the Deep Creek river sample. Boy was it a pain to clean. There was a lot of debris and silt. I bought a set of screen sieves a while back (http://www.ebay.com/itm/231111029804?_t ... EBIDX%3AIT) and used them to sieve out the plant material and larger debris. I only used the the three largest sieves since I think the smallest one would sieve out the largest diatoms. Even then the H2O2 boil had a bigger reaction than I thought it would and it bubbled up and out of my flasks and into my Pyrex catch pan. I tried to siphon up the spill and back into the boiling flask to save some of the material. Surprisingly when I added the dicromate, there was hardly any reaction, as well as the HCL boil went pretty smoothly. I tried getting a 'clean' diatom sample but it was impossible due to the large amount of fine mineral material. Even when I looked at a strew of the heavier bottom sand, which I was ready to throw out, showed some large diatoms...mostly Nitzschia, Pinnularia and Surirella. So, I kept it all...rocks and boulders, since I can pick out what I want and don't want to lose some nice large forms. The strews were really disappointing. They were very sparsly populated with diatoms. The dominate species seem to be the Nitzschia with a sprinkle of pinnularia and occasional surirella with some other nice centrics and pennates. I was also surprised at the mix of the salt water centrics in with the fresh water species. Deep Creek river does flow into the Chesapeake bay and is influenced by the tide so the water is somewhat brackish.

Pictures to follow when I have enough diatoms and diversity to show.

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Re: Doing Diatoms a Different Way

#111 Post by rnabholz » Wed Mar 01, 2017 4:16 pm

Hey Charles,

I was having flashbacks reading this post. Unpredictable reactions, boil overs, catch trays, gathering up the spill to save the sample, angst about straining out diatoms, high remaining mineral content, low diatom population, I certainly have all of those Merit Badges.... ;^)

It can be disappointing at times, but also very rewarding too, at least often enough to keep me coming back.

Thanks for posting, I am looking forward to the updates.

 

Rod
Last edited by rnabholz on Thu Mar 02, 2017 1:51 pm, edited 1 time in total.

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Re: Doing Diatoms a Different Way

#112 Post by Dale » Wed Mar 01, 2017 8:24 pm

Me too. All I have is a 10X loupe, for awhile.
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Re: Doing Diatoms a Different Way

#113 Post by KurtM » Thu Mar 02, 2017 3:28 am

I gotta start by saying Charles has a wonderful writing style, just love reading about his diatom trials and tribulations!

I guess all of us who have used the H202 protocol have had the delightful boil-over fun, as well as the mysterious hot/cold potassium dichromate reaction (or lack thereof). And we sure as heck know all about sediment and other debris fouling our strew mounts...

I can't help but believe that by comparing notes on our adventures, one day we'll see breakthroughs on some or all of these vexing little difficulties and arrive at a truly great method of cleaning and mounting diatoms simple enough for practically anyone to follow. We're already well along the way, since I think we can all agree that nobody was espousing cleaning protocols that were safe and accessible for those of us without training in chemistry and laboratory practice just a couple years ago.
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Re: Doing Diatoms a Different Way

#114 Post by Charles » Thu Mar 02, 2017 12:07 pm

Thanks guys!

Me thinks that 'dirtier' samples from river bottoms and vegetation will cause more intense reactions than 'cleaner' samples like from beach sand . I thought I was ready for the reaction, but then it started and then it just took off, especially when adding heat.

But I think we all do these things and posts, so others who want to attempt the same, will know what to expect.

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Re: Doing Diatoms a Different Way

#115 Post by rnabholz » Thu Mar 02, 2017 2:03 pm

I agree about the dirtier samples being the most energetic.

I started trying to manage these reactions in 15ml tubes, moved to 250ml Erlenmeyer flask, and now use a 500ml beaker. Last week I had a 70ml sample exit a 500ml beaker. 750 or 1000ml anyone?

It is really great to share our experiences, and I hope it encourages others to give it a try.

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Re: Doing Diatoms a Different Way

#116 Post by Charles » Fri Mar 03, 2017 12:09 pm

Pictures of the Deep Creek samples.

First is a typical strew slide with mainly rocks. You can see a Pinnularia and Nitzschia. Most fields had nothing but rocks.
1NS Field.jpg
1NS Field.jpg (152.91 KiB) Viewed 5588 times
The Storage slide with the most common forms found. A nice variety of forms considering the sparse population of diatoms per strew. The most common are the Nitzschia in the middle. These were at 3X setting on the B&L stereo.
1All Field 3X.jpg
1All Field 3X.jpg (152.21 KiB) Viewed 5588 times
Centrics with some nice large forms. I was surprised with the variety from a brackish river.
1Centrics.jpg
1Centrics.jpg (150.46 KiB) Viewed 5588 times
Pleurosira, Actinoptychus, different varieties of Eunotia and even some Hydrosera and Terpsinoe.
1Eunotia.jpg
1Eunotia.jpg (143.97 KiB) Viewed 5588 times
Some Pinnularia. Some of them are quite large.
1Pinnularia.jpg
1Pinnularia.jpg (152.52 KiB) Viewed 5588 times
Last edited by Charles on Fri Mar 03, 2017 3:17 pm, edited 3 times in total.

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Re: Doing Diatoms a Different Way

#117 Post by Charles » Fri Mar 03, 2017 12:22 pm

And more.

A nice varied collection of Surirella, including the twisting ones of Campylodiscus, Surirella spiralis and Entomoneis alata.
1Surirella.jpg
1Surirella.jpg (149.77 KiB) Viewed 5588 times
This is the Bone Yard...Broken diatoms, which I plan to mount to show internal structures. Also some of the broken ones, I have yet to find whole.
1Bone Yard.jpg
1Bone Yard.jpg (129.51 KiB) Viewed 5588 times
And then finally there are the UFOs. Spicules and other possible diatoms or not. I also found quite a few circular spheres; some clear, some matte and some black. I'm sure some if not all are the 'micro-balls' used in toothpaste and abrasives. But I would think these micro-ball spheres would float rather than sink into the muck and sand.
1UFOs.jpg
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Re: Doing Diatoms a Different Way

#118 Post by zzffnn » Fri Mar 03, 2017 1:53 pm

Your work paid off, Charles! Nice collection there.

This also shows that micro manipulation has definite advantage over strew with sandy samples.
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Re: Doing Diatoms a Different Way

#119 Post by rnabholz » Fri Mar 03, 2017 2:14 pm

Wow, a great mix of some very interesting stuff!

Trying to understand the scope of the work. When you make a strew for picking, do you cover the entire face of the slide? You obviously would not be concerned about matching the size of a cover slip.

How many strews does that collection represent?

I believe there is adhesive on the storage slide? How long can you expect it to hold the forms?

What approach do you take when making finished slides? Do you group by sample site, by form type, or are you the artistic arranger type?

Inspiring work Charles. Thanks very much for sharing it.

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Re: Doing Diatoms a Different Way

#120 Post by KurtM » Fri Mar 03, 2017 2:33 pm

Most excellent!

I find myself coming to a conclusion that brackish water offers the most diversity...
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