Doing Diatoms

Here you can discuss sample and specimen preparation issues.
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75RR
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Re: Doing Diatoms

#421 Post by 75RR » Mon Jan 02, 2017 1:39 pm

I don't have a way of measuring the accuracy of the device as I don't have a regulated device to test it against.
That is only because your lab is missing an essential must have "Mad scientist" item = oscilloscope ;)

https://www.youtube.com/watch?v=FZSNMz0m2Sw
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Re: Doing Diatoms

#422 Post by rnabholz » Mon Jan 02, 2017 3:19 pm

75RR wrote:
I don't have a way of measuring the accuracy of the device as I don't have a regulated device to test it against.
That is only because your lab is missing an essential must have "Mad scientist" item = oscilloscope ;)

https://www.youtube.com/watch?v=FZSNMz0m2Sw
Funny you should say that. About 10 years ago I bought a Hewlett Packard Oscilloscope at a garage sale. It was a huge, heavy suitcase style unit. I knew nothing about them or how to use it, but, as you say, thought I needed it for my secret lab. After tripping over it for many years, and having made no progress on learning how to use it, I got rid of it.......Dang, I knew it would come in handy some day.....

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Re: Doing Diatoms

#423 Post by Dale » Mon Jan 02, 2017 5:45 pm

The other essential is

http://www.cultofweird.com/science/diy-jacobs-ladder/


If you need any more ideas Jesse and Walter are working in the mega-meth lab in the
Breaking Bad marathon!
Dale
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Re: Doing Diatoms

#424 Post by rnabholz » Wed Jan 04, 2017 3:09 am

Another sample from Charles to process.

A a baggie of damp sand from the lower James River in Virginia.
James River Kit.jpg
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A small scoop into a rinse jar, add clean water and gently agitate
James River Scoop.jpg
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A bonus Stereo Scope object - just like the prize in Cracker Jacks!
James River Bonus.jpg
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Look at all of the goodies clouding the rinse water
James River Rinse.jpg
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I cleaned the entire sample and will let it all settle overnight. Looks promising....
James River Gold.jpg
James River Gold.jpg (121.12 KiB) Viewed 7370 times
Thanks Charles!

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Re: Doing Diatoms

#425 Post by Dale » Wed Jan 04, 2017 5:21 am

Rod, picture #4, if I took a tiny sample from just above the sand would it give
any hint as to its content? All the sand I collected from Ocean Shores has dried out.
Should I toss it? Sort of in a bind as all my stuff is already packed for the move, but the Nikon
and a well slide are accessible.
Dale
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Re: Doing Diatoms

#426 Post by rnabholz » Wed Jan 04, 2017 12:03 pm

Hi Dale,

Yes, your samples are still fine for our purposes. I would add some water to them, maybe let them set for a bit to allow for anything that might be stuck to free up, and then process like described above.

Once you have a batch like pic 4, take a sample, and take a look.

Good luck.

Rod

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Re: Doing Diatoms

#427 Post by rnabholz » Wed Jan 11, 2017 3:18 am

Situations like the sand sample as described a couple of entries up can be a bit tedious to prepare for chemical cleaning process.

Typically they don't yield a great amount of material, and it tends to lie under a good deal of water after the washing of the sand to extract it.

Taking that water off can be a challenge. You have to remove it without disturbing the diatoms settled at the bottom. I have used 3ml pipettes, that is a long, slow process. So I tried a turkey baster. More volume is good, but the difficulty with it is that you have to hold the bulb under tension as you place it in the water and position it for the water draw. The slightest twitch in your hand during that process can cause a puff of water to exit the baster and disturb the settled diatoms - now you get to wait a couple of hours for them to resettle.

There had to be a better way, so I went looking. I came across this on Ebay
Syringe Kit.jpg
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It is a 100ml syringe with tubing. I reasoned that the syringe would be a lot easier to control than that darn rubber ball at the end of the turkey baster. The control should be pretty darn precise with nearly zero chance of an inadvertent puff escaping.

To make it work I needed a way to position the tube under the water's surface, but above the settled diatom material - a clamp of some kind would do the trick. I am sure there is probably a specialized mechanism precisely for this purpose, likely with a long German name, like a Hoppenhauffer Clamp or something like that. Not having a Hoppenhauffer clamp handy, I had to get creative. So here is what I came up with - first the construction shot
Syringe Clamp Glue.jpg
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Two spring clothes pins, glued perpendicular to each other.

Looks like this when in use
Syringe Ready.jpg
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One clamps to the vessel, the other clamps to the tube. You can place the tube a precisely the depth you want by just sliding it up or down in the jaws of the second clothes pin.
Syringe Full.jpg
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When the syringe is full you can easily release the tube for emptying.

A careful and slow draw on the syringe pulls the water out without disturbing the settled diatoms, and a 100ml per fill, you can move a lot of water in a hurry, especially compared to 3 ml at a time with a pipette.

The syringe was $12 shipped, the clothes pins were free from the neighbors clothes line.......Just kidding, we had them laying around.

Thought some of you might find this useful.

Rod

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Re: Doing Diatoms

#428 Post by zzffnn » Wed Jan 11, 2017 3:57 am

Very nice, Rod.

Here is another idea. Use a rubber tube, the wider, the faster it sucks out water. Then shrink one end of tube around a glass/plastic dropper and use that to take out water. Place dropper higher up for now to prevent disturbing diatoms (neighbor's cloth pins can be used here too :mrgreen: ).

With the other end, just use your mouth to give it a quick suction. Take the tube out of mouth, before water gets into mouth. Then put the mouth end of the tube much lower than sample bottle, say under table. Water will drip out quickly by itself. Then just control depth of the dropper end.

You know early microbiologists used to use that kind of mouth pipette for moving pathogens. So one can get used to it.
Last edited by zzffnn on Wed Jan 11, 2017 2:51 pm, edited 1 time in total.

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Re: Doing Diatoms

#429 Post by rnabholz » Wed Jan 11, 2017 2:40 pm

Thanks zz,

I had considered the siphon approach, but the idea of a mouth full of swamp water or perhaps worse, Potassium Dichromate ruled that out for me.

I can still taste gasoline from an incident many years ago...

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Re: Doing Diatoms

#430 Post by Charles » Wed Jan 11, 2017 4:42 pm

Great idea Rod!

It's amazing how we all think somewhat alike. I too was tired of siphoning off the large amounts of fluids from beakers after settling using the disposable pipettes, and after rummaging through my wife's kitchen drawers found some 50ml syringes used for injecting seasoning/marinate into turkeys before roasting. Each seasoning/marinate package (this is the one she buys:https://www.walmart.com/ip/10313897?wml ... 3=&veh=sem) she bought the last 3-4 years comes with its own syringe with a large bore needle, so she had accumulated 3 or 4 of these and I took one for my needs. The pointed needle part was beveled with a couple round holes near the tip, so I just clipped the pointie end off and use the rest of the needle on the syringe. The whole package with marinate and syringe is less than $4. Now I can syringe out 40-50ml of liquid at a time, which is a great time saver.

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Re: Doing Diatoms

#431 Post by rnabholz » Wed Jan 11, 2017 7:26 pm

Excellent Charles, even better, no out of pocket expense.

It really does speed things up a great deal. Well worth it!

Thanks

Rod

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Re: Doing Diatoms

#432 Post by rnabholz » Wed Feb 01, 2017 12:54 am

Well Charles started the true confessions with his story about the errant needle scrambling his arranged slide and subsequent issues with his needles. My Turn.....

All day I was thinking what a great evening it would be. I had finished four slides from a sample that Charles had sent me. They were just waiting for me to come home, throw them on the scope and do some slide surfing on what promised to be some interesting marine forms, maybe shoot some pics to share, should be great fun.

Home, quick supper and downstairs to get started. Went to get the slides where I had left them to cool the night before, sitting in place on the switched off hot plate, and there they were......
Toasted DOH.jpg
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Some dummy forgot to shut down the hot plate and they had been cooking for about 26 hours.... DOH!

I remember thinking last night that I wanted to let them cook about 10 minutes more - and apparently that was the last thought I had on the subject.

Soooooo - why don't we try that again, maybe shorten the cooking time by say, 25.5 hours or so.

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Re: Doing Diatoms

#433 Post by Charles » Sun Feb 05, 2017 5:34 pm

Oh no! I'm always paranoid of leaving the hot plate on with something on it but have overcooked slides a few times.

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Re: Doing Diatoms

#434 Post by KurtM » Sun Feb 05, 2017 6:11 pm

For some reason I missed Rod's hot plate hiccup when it was first posted. Man, those slides are really fried, aren't they?

Well, I am certainly very much in the club of those who can see forgetting to turn the hot plate off coming a mile away. So here's what I've devised: an extendable lamp positioned over the hot plate, and dedicated 100% to it. Cardinal Rule in my lab: if I turn on the hot plate, I also turn on the lamp; conversely, when I turn off the hot plate, so goes the lamp. Period. In this way I have yet to go to bed leaving the hot plate going all night. A fringe benefit is whatever I put on the hot plate is well illuminated and easy to see.
Cheers,
Kurt Maurer
League City, Texas
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Re: Doing Diatoms

#435 Post by rnabholz » Sun Feb 05, 2017 7:06 pm

Yes, they are quite well done.

This is the first time this has happened to me. I think I will blame it on the extended cook time of the inverted cure process. After inverting, I let them cure for about 20 minutes at high temp and then shut the plate down and let them cool in place.

I usually set this to remind me about such things
IMG_20160715_180210-1024x760.jpg
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But failed to do so this time apparently....

Your light idea is a good one.

Maybe an ankle high trip line on the stairs...Gotta get my attention.

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Re: Doing Diatoms

#436 Post by rnabholz » Fri Feb 10, 2017 4:53 am

Properly baked, frosted and decorated... ;^)
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Re: Doing Diatoms

#437 Post by Charles » Mon Feb 13, 2017 1:37 am

Yes, they look perfect and delicious!

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Re: Doing Diatoms

#438 Post by KurtM » Mon Feb 13, 2017 1:50 am

Dang, you need a label printer! I'm getting carpal tunnel syndrome just looking at your picture. :P
Cheers,
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Re: Doing Diatoms

#439 Post by rnabholz » Mon Feb 13, 2017 4:06 am

Charles wrote:Yes, they look perfect and delicious!
Not perfect, but delicious, yes. Thanks Charles.

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Re: Doing Diatoms

#440 Post by rnabholz » Mon Feb 13, 2017 4:09 am

KurtM wrote:Dang, you need a label printer! I'm getting carpal tunnel syndrome just looking at your picture. :P
I admit that printing the labels is my least favorite part of the process, but it always takes less time than my mind leads me to think it will.

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Re: Doing Diatoms

#441 Post by Dale » Mon Feb 13, 2017 11:44 pm

Those beautiful rings! Could I use the same enamel to build up a perimeter
dam to create a very small rectangular well slide? 2mm thickness.
Dale.
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Re: Doing Diatoms

#442 Post by zzffnn » Tue Feb 14, 2017 12:48 am

Dale wrote:Those beautiful rings! Could I use the same enamel to build up a perimeter
dam to create a very small rectangular well slide? 2mm thickness.
Dale.
Dale,

Rod will tell you for sure. I doubt that enamel is strong enough to be such a high dam though. Maybe 0.5-1 mm, but probably not 2 mm. Some enamel can be dissolved by alcohol.......

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Re: Doing Diatoms

#443 Post by KurtM » Tue Feb 14, 2017 1:47 am

Testor's model enamel wouldn't be suitable for the purpose of building a dam. It'd take forever if you ever managed it at all. If it were me I'd think along the lines of a turntable and UV cure acrylic. Or part rings from aluminum tubing on a lathe. Or get a circle cutter and make foil or paper rings. Round things are often easier to make than square things, counter-intuitive as it may be.

Circle cutter: http://tinyurl.com/zdeeajv
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Kurt Maurer
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Re: Doing Diatoms

#444 Post by rnabholz » Tue Feb 14, 2017 3:40 am

Hi Dale,

You have gotten good advice regarding the Testors Enamel, it just doesn't pile up much, so getting any sort of height to build isn't likely to be easy.

Rod

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Re: Doing Diatoms

#445 Post by Dale » Tue Feb 14, 2017 3:49 am

Fan, Kurt, and Rod, thank you all for saving me from a futile attempt.
Dale
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Re: Doing Diatoms

#446 Post by zzffnn » Tue Feb 14, 2017 4:06 am

Dale,

This is what I use:
Many credit cards are 0.7mm-1mm thick. You can cut them to form walls of your dam. Then simply use silicone glue (from say HomeDepot) to glue them (then wait about 24 hrs to let the glue set). Such a well slide will last maybe 5 uses, if you are not very careful, after which the walls may get loose and you would need to glue them again. Still easy and cheap to make/repair.

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Re: Doing Diatoms

#447 Post by Dale » Tue Feb 14, 2017 4:54 am

I like that, it's on my hot projects list now. Thanks, (and thanks).
Dale
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Re: Doing Diatoms

#448 Post by rnabholz » Thu Mar 02, 2017 2:14 am

I have recently been experimenting with a "No Spin" approach to cleaning diatoms. On Charles' great thread "Doing Diatoms Differently" I outlined the idea and the reasoning. I trust Charles won't mind if I borrow some of the verbiage that I typed in that thread to save me typing it again here:

"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 supernatent 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 therefore 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 here is a look at the process in action.
Start
Start
No Spin Protocol Start.jpg (153.96 KiB) Viewed 6904 times
Here you see the sample as it appears after the Hydrogen Peroxide and Potassium Dichromate have reacted, the extra chemicals have been removed and it has been transferred to the 500ml Graduated Cylinder.
No Spin Protocol 1st Dilution.jpg
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Here you see that sample with water added to a total volume of 500ml. Obviously intensity of the chemical's color has been diminished reflecting the dilution.
No Spin Protocol Suspension.jpg
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Here you see the sample suspended in the cylinder immediately after the dilution.
No Spin Protocol Settled.jpg
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The sample took around 20 to 30 minutes to settle properly, but of course that does not require close supervision, so I tended to other matters while that happened.
No Spin Protocol Suspension Removal.jpg
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Once it was settled, I used the syringe and tubing to carefully remove the supernatent.

Continued...

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Re: Doing Diatoms

#449 Post by rnabholz » Thu Mar 02, 2017 2:35 am

From there it was just like shampooing your hair "Rinse and Repeat"
No Spin Protocol 2nd Suspension.jpg
No Spin Protocol 2nd Suspension.jpg (160.38 KiB) Viewed 6903 times
Here is the second suspension of the sample - note the color change.

And the sample settled after the second suspension.

I did one more suspension just to be on the safe side and the results were a nice clean sample.

So what conclusions can be drawn?

1. In the end, I don't think it decreased the time it took to go from start to finish vs the more conventional small tube centrifugation. BUT, the big advantage is that most of the time that this process took could elapse unattended, while with the Small Tube Spin process required constant involvement, with 5 minute spins, clearing of rinse water, pipetting to unpack the sample in the bottom of the tube, all of that across multiple tubes, over 5 to 6 cycles in order the get them clean with the limited space for rinse water in the small tubes. The big tube could just be left to settle unattended for 20 to 30 minutes (or longer).

2. My impression having done two samples with this protocol is that it is gentler. I see more complete undamaged frustules in these samples that had been my experience with centrifuged samples. I HAVE NOT done a direct comparison processing the same sample using both methods and comparing. Perhaps this summer when samples are more plentiful I can do that test.

3. A centrifuge is an option, but not a requirement for processing diatoms.

4. The only issue I have run across has to do with the attraction of diatoms to the sides of the cylinder. There seems to be a tendency for some of the sample to settle on the side of the cylinder and not fall to the bottom. Not sure as to the cause of this, I see it in the 15ml tubes as well, but centrifugation takes care of the issue. I don't believe that it would capture all of any specific type of form, but there is some possibility of loss of some diatoms as a result of this issue. If your sample is relatively small, it may be something you should consider before using this method as you might find the loss to be potentially too great to risk. With decent sized samples, I don't feel that it would be an issue.

So that is what I know about this approach so far. I plan to continue to use it as I find it convenient and productive. I will update as I have news to report.

Thanks for reading.

Rod
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Re: Doing Diatoms

#450 Post by KurtM » Thu Mar 02, 2017 3:05 am

This is great stuff. Rod's methods diverge from mine, but it's a grand thing because it means there are two paths being explored, and I couldn't care less who the trailblazer is -- I'm intensely interested in finding the best route from A to B, however, and may the best method win!

I will add this for general interest: I find there's a big difference between centrifugation action in the electric machines versus the hand cranked variety. I would have thought that spinning is spinning, and the means of making the buckets rotate could have nothing to do with the results. But what do I know? The original Youtube video (link below) suggested 3 minutes in electric centrifuge or 1 minute in a hand cranked one, and my own experience is consistently that five minutes in my electric centrifuge barely accomplishes what 45 seconds does neatly in my hand cranker.

Ref: https://www.youtube.com/watch?v=l-uN2RPvDSM
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Kurt Maurer
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