Doing Diatoms

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rnabholz
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Re: Doing Diatoms

#61 Post by rnabholz » Tue May 24, 2016 1:30 pm

That is fantastic information Kurt! Thank you.

Armed with this new knowledge, I think I will be much more relaxed in the next session!

The idea of thinning on the fly is a real revelation to me. I was wondering how I would ever hit upon the correct balance mixing up batches. Can't wait to try again.

Incredible work on the signs. I can't imagine doing that all freehand. That is a serious talent you have there.

Regarding Iowa diatoms, it is possible that you may have some in your future....
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Re: Doing Diatoms

#62 Post by KurtM » Tue May 24, 2016 3:02 pm

I got to thinking about this a little more, and had to start shaking my head at you. When I first tried ringing slides, there was no difficulty whatever in grabbing worthless ones to experiment on, that would be no trouble to ruin and throw away. If every permanent slide in your lab is a keeper, then I must resist the temptation to drown myself next time I'm out collecting...

Here's another tip: plastic rinse bottles from the lab are perfect for delivering small amounts of mineral spirits. Like this: eBay item number: 171909229036.
Cheers,
Kurt Maurer
League City, Texas
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Re: Doing Diatoms

#63 Post by rnabholz » Tue May 24, 2016 4:49 pm

KurtM wrote:I got to thinking about this a little more, and had to start shaking my head at you. When I first tried ringing slides, there was no difficulty whatever in grabbing worthless ones to experiment on, that would be no trouble to ruin and throw away. If every permanent slide in your lab is a keeper, then I must resist the temptation to drown myself next time I'm out collecting...

Here's another tip: plastic rinse bottles from the lab are perfect for delivering small amounts of mineral spirits. Like this: eBay item number: 171909229036.
I suspect the issue here is low standards combined with unchecked sentimentality.

How could my babies be worthless? Even if they are so dense light will barely pass? The bad man says you are worthless, no you aren't......

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Re: Doing Diatoms

#64 Post by rnabholz » Wed May 25, 2016 1:52 am

The three latest samples are clean and ready to mount. My batches never seem to get to that super white color that I have seen in the videos, remaining instead a light yellow color. Examining them under the scope in a wet mount shows that they are clean and so I am calling it good to go.

I decided to make 4 slides of just one of the samples the goal for the evening. I selected the sample from the park in Fairbank that showed such great variety.

I have found that for my intended purpose of photographing the diatoms that I prefer a sparser density to provide separation between potential subjects. My early slides were a bit dense to say the least. So to start I place a small sample taken with a pipette, trying my best to take just the top layer of the material in the tube as the denser sand had most likely settled to the bottom of the tube. It looked like this
May24 Batch Sample.jpg
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If I were to place that on the slide, it would be a mess of tangled and stacked diatoms everywhere. So I add distilled water to dilute the sample until it takes on a cloudy appearance.
May24 Batch Dilution.jpg
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Next we need cover slips. I wanted to mention a couple of things quickly. I have read references that recommend that the cover slips used be as close as possible to the .17mm thickness including the thickness of the mountant. While I have not actually done any measurements on the thickness of the mountant, I reasoned that if I selected cover slips of around .1675mm that should put me in the ball park. To that end, I took the time to measure my slips with a micrometer and sort them to the nearest .01mm. Angels dancing on the head of a pin? Maybe, but at least I can check that off of the list of potential issues.

The next tip I owe to Kurt. He told me about this little device and how it made his life easier with regard to handling cover slips, and by golly he was right. For $2 shipped from China (how do they DO that?) I got my very own Suction Pen. It really is handy when it comes to picking up slips, especially when you have cleaned them and have them ready to go. Thanks Kurt.
May24 Batch Suction Pen.jpg
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Four Sorted and Sized slips go on the hot plate set to about 120 F. I gently agitate the tube containing the diluted sample and draw a few drops out with a pipette and place it on a slip. The same process for the other three slips.

Next I set out four cleaned slides on my template sheet. Then grab the Pleurax, and using a toothpick place a drop on the slide over the center mark of the template.
May24 Batch Loaded Template.jpg
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I make a effort to make the drop a bit bigger than the last time as I had issues with covering all of the slip with a couple of slides the last time
May24 Batch Suction Pluerax Drop.jpg
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By now the slips are dry and the diatoms are fixed in place on the slips. Now using a tweezer, I take the slip and place it diatom side down onto the Pleurax drop, and that use the tweezer to make any adjustments to its position to center it on the template.

After all four are done, I transfer the slides one by one back to the hot plate, and turn up the heat to 350F. Once the plate reaches that temp, the Pluerax bubbles and the solvent evaporates. I give it 15 minutes at the target temp and take the off the plate to cool.
Last edited by rnabholz on Wed May 25, 2016 12:00 pm, edited 1 time in total.

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Re: Doing Diatoms

#65 Post by rnabholz » Wed May 25, 2016 2:13 am

The group came out very well this time. Good coverage of the Pleurax under the cover slips and very little spatter to clean up.
May24 Batch Finished Batch.jpg
May24 Batch Finished Batch.jpg (37.91 KiB) Viewed 3091 times
Now just a short wait and I can load one up on the stage and take a look!

As I lean into the eyepieces, I can see that the density is just about as I had hoped, a nice compromise between spacing with yet enough in each field to keep the eye busy.

I turn the 20x into position and start scanning. The first thing I notice is a conspicuous absence of many of the larger forms I had seen in the live sample. A closer look shows a good deal of bits and shards that likely explain the missing forms. I review all four slides and all show the same issue.

That is disappointing to say the least. But I decide to turn up the power, first to the 40x and ultimately to the 100x. While there is a lot missing, at the higher powers there is still a good deal to see. The smaller forms do not show the damage done to the larger ones.

Now curious about the other two batches waiting in the wings, I made a wet slide with a sample from both, and unfortunately it would appear that they suffered the same fate.

So what went wrong? Here are my theories.

1. I tried to process too large a sample. The effect of this was inefficient cleaning by the chemicals, the result of which was multiple rounds of cleaning being necessary

2. I used more heat during this round. In order to improve the efficiency of the cleaning (see #1 above) I got more aggressive with the temperature of the hot water bath.

The dumb part is that it just doesn't take very much material to make a whole lot of slides, so why the heck did I try to do such large batches? 'Cause if a little bit is good, a whole lot is much better right? - not in this case.

Smaller batches in the tubes means that the chemicals can infuse throughout the material more efficiently and do their job.

Soooooo, I think I will start a new set of batches tomorrow, and resist the urge to Super Size it.

But while they cook, I will get the camera out and shoot some of the forms on the slides. I saw at least a half dozen forms that I had never seen before, tiny though they were, they are still so cool.....

Thanks for reading.

Rod

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Re: Doing Diatoms

#66 Post by zzffnn » Wed May 25, 2016 3:24 am

Rod,

Is heating mounted sample/Pleurax at 350F for 15 min necessary? That may be your most aggressive heating, more so than hot water bath, at least temperature wise. If you suspect toouch heating caused big diatoms to break, then you might want to reduce heat there.

Also, you may want to reduce centrifugation force (lower rpm). Since you have enough end yield, you don't need to spin down everything that hard. Diatoms are more dense than most other protists. You probably don't need more than 1200 rpm.

Also, try to pipette less and gently. Shear force can break silica pieces.

I am just guessing here. I don't have any diatom clenaing./mounting experience. But I did work in research labs for around 10 years and some DNA/protein/bacteria/cell isolation procedures are highly similar to diatom cleaning.

The best way to find out what/which step destroyed the big diatoms, is to take a sample before and after each and every procedure, and view/record/compare images of those samples. If you have intact big diatoms before procedure X, and lost them after X, then X is the fault.
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Re: Doing Diatoms

#67 Post by KurtM » Wed May 25, 2016 3:43 am

Ha ha - it's vaguely therapeutic to see I'm not the only one who develops personal relationships with inanimate objects! Guess I begin to take a dim view of some of my slides because they refuse to cooperate, and/or live up to my haughty expectations. Oh, so that's the way you wanna be, eh? Well take that you dumb slide!

You seem to take a more methodical (fussy and worried) approach to all this than I, and the net result is bound to be much improvement on both our parts, so good on you. For instance, I also have a lot of mangled remains of once proud forms in my samples, but never thought enough of it to even mention it, let alone regard it as a problem to solve - I'm just happy to be getting halfway decent results at all. Well if you're going to get all tree hugger and "Save The Diatoms", then two can play that game! We both know this isn't supposed to be a contest, but it dang sure is, and we're both sure to win if we keep it up.

Now all we need is another victim to get hooked on this silliness to really jack up the stakes. Come on Fan, you know you want to clean some diatoms. Resistance is futile, you will be assimilated. :twisted:
Cheers,
Kurt Maurer
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Re: Doing Diatoms

#68 Post by rnabholz » Wed May 25, 2016 4:12 am

zzffnn wrote:Rod,

Is heating mounted sample/Pleurax at 350F for 15 min necessary? That may be your most aggressive heating, more so than hot water bath, at least temperature wise. If you suspect toouch heating caused big diatoms to break, then you might want to reduce heat there.

Also, you may want to reduce centrifugation force (lower rpm). Since you have enough end yield, you don't need to spin down everything that hard. Diatoms are more dense than most other protists. You probably don't need more than 1200 rpm.

Also, try to pipette less and gently. Shear force can break silica pieces.

I am just guessing here. I don't have any diatom clenaing./mounting experience. But I did work in research labs for around 10 years and some DNA/protein/bacteria/cell isolation procedures are highly similar to diatom cleaning.

The best way to find out what/which step destroyed the big diatoms, is to take a sample before and after each and every procedure, and view/record/compare images of those samples. If you have intact big diatoms before procedure X, and lost them after X, then X is the fault.

Hey zz

The 350f for 15 minutes was the recommendation of the manufacturer/supplier of the Pleurax, so it think that is less likely the cause.

In support of that, I would think if they were breaking during the curing, I would see the bits of a single form in close proximity. My impression is that it is a stew of bits, well mixed.

I certainly can slow down the centrifuge, I have been running it at 3000 rpm, certainly an opportunity to go slower.

I do have a successful run to reference, and the key differences were the batch size, the rounds and duration of the HP soaks and the heat of the bath. I think that I will go back to the process that worked, and add in your excellent suggestions with regard to the physical handling.

Thanks for the ideas.

Rod
Last edited by rnabholz on Wed May 25, 2016 12:35 pm, edited 1 time in total.

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Re: Doing Diatoms

#69 Post by rnabholz » Wed May 25, 2016 4:24 am

KurtM wrote:Ha ha - it's vaguely therapeutic to see I'm not the only one who develops personal relationships with inanimate objects! Guess I begin to take a dim view of some of my slides because they refuse to cooperate, and/or live up to my haughty expectations. Oh, so that's the way you wanna be, eh? Well take that you dumb slide!

You seem to take a more methodical (fussy and worried) approach to all this than I, and the net result is bound to be much improvement on both our parts, so good on you. For instance, I also have a lot of mangled remains of once proud forms in my samples, but never thought enough of it to even mention it, let alone regard it as a problem to solve - I'm just happy to be getting halfway decent results at all. Well if you're going to get all tree hugger and "Save The Diatoms", then two can play that game! We both know this isn't supposed to be a contest, but it dang sure is, and we're both sure to win if we keep it up.

Now all we need is another victim to get hooked on this silliness to really jack up the stakes. Come on Fan, you know you want to clean some diatoms. Resistance is futile, you will be assimilated. :twisted:

I won't lay awake tonight about those lost big forms, but dang it, those are the easiest to photograph! Those tiny ones are hard!

The missing forms really struck me on the first look because the remaining large ones were so limited in variety. I guess that is what bothers me the most, as I am still "collecting" forms and kind of feel that this is a missed opportunity.

The good news is I gots lots of live sample left and can go again until I get it right.

I agree that having someone to compare notes with is fantastic. We should be Pro Diatom Wranglers in no time at all.
Last edited by rnabholz on Wed May 25, 2016 12:37 pm, edited 1 time in total.

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Re: Doing Diatoms

#70 Post by mrsonchus » Wed May 25, 2016 9:32 am

Great adventures Gents - game-on I'd say. My money's on the shear of excessively-high rpm centrifuge stage/s if I have to guess (as I'm poking my snout into an area I know nothing at all about! :D )....
It's great to follow the process that's evolving as you hone your skills with these glassy-devils! :D

Keep it up - we love it. :)

something to dream about.....micromanipulators..... you know you must..... :D
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Re: Doing Diatoms

#71 Post by Charles » Wed May 25, 2016 12:25 pm

KurtM wrote: Now all we need is another victim to get hooked on this silliness to really jack up the stakes. :
I've been watching you guys and I've stuck my toe in the waters a couple years back...maybe it's time to wade back in. I've got a hot plate, centrifuge, test tubes, Pleurax and Zrax, some chemicals...just need to clear a space on my work bench and some time.

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Re: Doing Diatoms

#72 Post by zzffnn » Wed May 25, 2016 12:36 pm

And the Diatom Hunter Club is formed!

I will join too, though I don't currently have space for those lab equipments. Maybe 10 years down the road. I will happily buy mounted slides from you guys though.

Rod,
If you want to be safe with centrifuge speed, may I suggest around 600 rpm. That is the standard speed that we used in labs for human cells (which are less dense than diatoms and more delicate). You may compare water mounts of harvests after 600 rpm vs 3000 rpm, looking at big diatoms.
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Re: Doing Diatoms

#73 Post by rnabholz » Wed May 25, 2016 1:10 pm

mrsonchus wrote:Great adventures Gents - game-on I'd say. My money's on the shear of excessively-high rpm centrifuge stage/s if I have to guess (as I'm poking my snout into an area I know nothing at all about! :D )....
It's great to follow the process that's evolving as you hone your skills with these glassy-devils! :D

Keep it up - we love it. :)

something to dream about.....micromanipulators..... you know you must..... :D
Thanks John.

I can barely get these strews on the slide, and you want me to arrange individuals into fanciful shapes? If I were ever to try it, I would try to spell out the words" Nervous Breakdown", and I would bet that the doctors would find only the first half of the letter "N"......

Glad you are enjoying the thread.

Rod

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Re: Doing Diatoms

#74 Post by rnabholz » Wed May 25, 2016 1:13 pm

Charles wrote:
KurtM wrote: Now all we need is another victim to get hooked on this silliness to really jack up the stakes. :
I've been watching you guys and I've stuck my toe in the waters a couple years back...maybe it's time to wade back in. I've got a hot plate, centrifuge, test tubes, Pleurax and Zrax, some chemicals...just need to clear a space on my work bench and some time.
Charles, You are all set! Come on in, the water's fine and full of slimy rocks!

Join the fun, the more the merrier, and Kurt and I could use an expert to learn from.

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Re: Doing Diatoms

#75 Post by rnabholz » Wed May 25, 2016 1:19 pm

zzffnn wrote:And the Diatom Hunter Club is formed!

I will join too, though I don't currently have space for those lab equipments. Maybe 10 years down the road. I will happily buy mounted slides from you guys though.

Rod,
If you want to be safe with centrifuge speed, may I suggest around 600 rpm. That is the standard speed that we used in labs for human cells (which are less dense than diatoms and more delicate). You may compare water mounts of harvests after 600 rpm vs 3000 rpm, looking at big diatoms.
Thanks zz

Let me ask another question relating to the centrifuge. I usually use 5 minutes as the standard duration of my spins, but as my centrifuge does not have a timer, and I get distracted with other tasks, they often go longer. Can that be detrimental?

I have a kitchen timer I can use if it is a critical issue.

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Re: Doing Diatoms

#76 Post by zzffnn » Wed May 25, 2016 1:34 pm

Rod,

At 600 rpm, longer time should not be directly detrimental. Not sure about 3000 rpm.

Indirectly:
longer spin time will compact the precipitation further, thus necessitating more pipetting in the following wash step. More pipetting may shear big diatoms. Loose precipitation can be washed by gently inverting tubes.
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Re: Doing Diatoms

#77 Post by Charles » Wed May 25, 2016 2:37 pm

I am by no means an expert, just want to learn like you two are doing and add some samples from the Common Wealth of Virginia. :D

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Re: Doing Diatoms

#78 Post by rnabholz » Wed May 25, 2016 2:42 pm

Charles wrote:I am by no means an expert, just want to learn like you two are doing and add some samples from the Common Wealth of Virginia. :D
A great addition to the mix!

It occurs to me that we now need to add a West Coaster to cover all of the cardinal points of the compass.

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Re: Doing Diatoms

#79 Post by KurtM » Wed May 25, 2016 2:46 pm

This has me wanting to do a series of centrifuge experiments. But instead of meticulously and tediously checking each result under the scope, instead simply see what minimum requirements are for settling and decanting. Also be interesting to see how electric and hand operated centrifuges compare. I can see where minimum packing would mean minimum "unpacking", and I suspect Fan is on to something when he accuses the pipet of doing damage.

I have already thought of micromanipulation, which intrigues me quite a lot, actually. I got a kick out of Rod's fear of a nervous breakdown though, ha ha! When painting signs and getting into really intensely precise brush work that must appear easy and flowing to end up looking right, I often wonder if I'm finding it relaxing or terrifyingly agonizing...? Nervous breakdown ... yeah, I can see that...

Charles, I hope you do join in! Not only should it be great fun to see what you come up with in the way of posts given your wonderful stash of stuff, but I'd also be eager to see what forms you come up with.
Cheers,
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Re: Doing Diatoms

#80 Post by rnabholz » Wed May 25, 2016 3:15 pm

I had forgotten that I snapped an afocal, handheld cellphone shot of one of the fields last night.

This one contains what I believe is Kurt's new friend from yesterday, Nitzschia, as well as, at its 10 o'clock, another new one for me which I believe is Aneumatis family, possibly Pseudotusculus. These are small, around 15 microns or so.

Kind of cool that Kurt and I turned up the same form for the first time the same day, a thousand miles apart....

Here is the snapshot, 100x, edited on my phone.
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Re: Doing Diatoms

#81 Post by rnabholz » Wed May 25, 2016 4:32 pm

KurtM wrote:This has me wanting to do a series of centrifuge experiments. But instead of meticulously and tediously checking each result under the scope, instead simply see what minimum requirements are for settling and decanting. Also be interesting to see how electric and hand operated centrifuges compare. I can see where minimum packing would mean minimum "unpacking", and I suspect Fan is on to something when he accuses the pipet of doing damage.
I ran a rinse spin at 1800 once, and saw a bit of cloudiness in the supernatant. So in my irrational desire to capture those 1000 suspended frustules along with the other 50,000 snuggled down in the bottom of the tube, I ruled out that speed as a usable setting.....DOH!!

I will certainly revisit that, and lower speeds, and happily trade a few individuals for a better preserved catch.

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Re: Doing Diatoms

#82 Post by zzffnn » Wed May 25, 2016 5:14 pm

rnabholz wrote:
I ran a rinse spin at 1800 once, and saw a bit of cloudiness in the supernatant. So in my irrational desire to capture those 1000 suspended frustules along with the other 50,000 snuggled down in the bottom of the tube, I ruled out that speed as a usable setting.....DOH!!

I will certainly revisit that, and lower speeds, and happily trade a few individuals for a better preserved catch.
Run a water mount under scope and see what that cloudiness is? They may not be diatoms. More likely organic debris smaller than bacteria.

800rpm would spin down most human cells, whose density is much less diatoms. So you may not be losing any diatom. 1800rpm will rupture many human cells or ciliates.
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Re: Doing Diatoms

#83 Post by Charles » Wed May 25, 2016 5:22 pm

[quote="rnabholz"

It occurs to me that we now need to add a West Coaster to cover all of the cardinal points of the compass.[/quote]

Yes, and also from England, Europe, Australia, Russia...

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Re: Doing Diatoms

#84 Post by rnabholz » Wed May 25, 2016 6:27 pm

Charles wrote:[quote="rnabholz"

It occurs to me that we now need to add a West Coaster to cover all of the cardinal points of the compass.
Yes, and also from England, Europe, Australia, Russia...[/quote]

Absolutely! Diatoms Without Borders!

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Re: Doing Diatoms

#85 Post by mrsonchus » Wed May 25, 2016 7:01 pm

Uh-oh, I must resist your silky-silica and turn away from your fabulous frustrating frustules - before I find myself adding a UK arm! :D Great to follow, but it's leaves and stems for me! :D
Keep up the good work brave-fellows, very interesting indeed, and very impressive progress by all. Some really good work happening here, you're creating a 'collective momentum' I think, it will lead to great things! :D

Thanks all for the super thread. :)
John B

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Re: Doing Diatoms

#86 Post by KurtM » Wed May 25, 2016 11:11 pm

zzffnn wrote:Run a water mount under scope and see what that cloudiness is? They may not be diatoms.
YES! You have a microscope sitting right there, so use it! :idea:

Makes me want to repeat one of my favorite microscopy stories. A former rolling student turned buddy in Florida sent a sand sample from a half-submerged sand bar near St James City, Florida that he gathered for me while out kayaking. I washed the sand before examining it, and no sooner finished that step than began wondering what, exactly, I had washed away? So I got a bit more, washed it, but retained the rinse water. The "dirt" I had seen turned out to be diatoms, and they made up the most spectacular sample I have ever had, bar none! Rod, you have a slide of that material in the set I sent you. Boy, am I ever glad it occurred to me to have a look at that stuff, and boy, do I ever love having a microscope handy!

mrsonchus, we forgive you. The world needs botany enthusiasts too! 8-)
Cheers,
Kurt Maurer
League City, Texas
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Re: Doing Diatoms

#87 Post by rnabholz » Thu May 26, 2016 2:52 am

KurtM wrote:
zzffnn wrote:Run a water mount under scope and see what that cloudiness is? They may not be diatoms.
YES! You have a microscope sitting right there, so use it! :idea:

Makes me want to repeat one of my favorite microscopy stories. A former rolling student turned buddy in Florida sent a sand sample from a half-submerged sand bar near St James City, Florida that he gathered for me while out kayaking. I washed the sand before examining it, and no sooner finished that step than began wondering what, exactly, I had washed away? So I got a bit more, washed it, but retained the rinse water. The "dirt" I had seen turned out to be diatoms, and they made up the most spectacular sample I have ever had, bar none! Rod, you have a slide of that material in the set I sent you. Boy, am I ever glad it occurred to me to have a look at that stuff, and boy, do I ever love having a microscope handy!

mrsonchus, we forgive you. The world needs botany enthusiasts too! 8-)
Frankly, I was so sure they were diatoms, that it never occurred to me to check...lesson learned

Yes, the sole marine slide in my fledgling collection. Some cool stuff on there.

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rnabholz
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Re: Doing Diatoms

#88 Post by rnabholz » Thu May 26, 2016 2:58 am

I managed to shoot a few photos of the #1 slide of the Fairbank batch tonight. Still lots of interesting things to see on them, even if the larger forms are missing.

I posted them in Pictures here:. viewtopic.php?f=6&t=3104

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Re: Doing Diatoms

#89 Post by rnabholz » Fri May 27, 2016 12:23 am

I thought some of you may be interested in what effect the amount of dilution I used, as described above, had on the views.

Below are shots taken of the same field, at 10,20,40 and 100x. They are representative of the views found across the slides. As you can see, there is pretty decent separation, but lots to look in every view. For photography it is a pretty good balance, isolated subjects make for good image making options.

These were quick shots taken afocally with the phone, so I apologize for any issues, but I think the tell the story.
Dilution Field 10x.JPG
Dilution Field 10x.JPG (83.38 KiB) Viewed 2927 times
Dilution Field 20x.JPG
Dilution Field 20x.JPG (100.53 KiB) Viewed 2927 times
40x
40x
Dilution Field 40x.JPG (70.43 KiB) Viewed 2927 times
100x
100x
Dilution Field 100x.JPG (75.47 KiB) Viewed 2927 times

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Re: Doing Diatoms

#90 Post by zzffnn » Fri May 27, 2016 12:41 am

Very nice work, Rod. That dilution worked perfectly, to my eyes.

Those diatoms there are not too small. Those even larger slender ones are very easy to break, I have broken quite a few when I broken my Klaus Kemp slides. Large slender ones, such as Gyrosigma, always break first.

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