Doing Diatoms

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rnabholz
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Re: Doing Diatoms

#391 Post by rnabholz » Tue Dec 06, 2016 4:30 pm

Regarding centrifuge speeds.

The manual for my centrifuge gives speed ranges for specific dial settings with specific rotors. In this case for the 8 position horizontal bucket rotor, it tells me that a setting if 36 on the speed dial makes 1000 rpm. I have been running it at around 15 which i think puts me in the 400rpm range.

That assumes that the motor is running up to the standard of the testing unit, that there is perfect linearity of the power curve of the speed control, and it is not a full moon in a month with the letter "r" in it......

So I am not exactly sure, but here is a cheap way to find out:

AGPtek® Professional Digital Laser Photo Tachometer Non Contact RPM Tach https://www.amazon.com/dp/B004Q8L894/re ... rybGH84ZE8

Looks like a great stocking stuffer....

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Re: Doing Diatoms

#392 Post by rnabholz » Tue Dec 06, 2016 4:38 pm

Charles wrote:Looks like some good stuff Rod! Why use distilled water and not just tap water for the initial suspension and cleaning and then use the DW on the final 3 rinses after it has all been cleaned and rinsed 3 times in tap water first?

I've given up on the centrifuge. I just let it settle by gravity for about 2 hours, I then siphon the top water off to another container which I let stand even longer in case there are diatoms still floating around. May take longer but I know the forms won't be damaged.

Keep up the good work!
Thanks Charles.

Your point about the distilled water is a good one, tap water will absolutely work fine for those steps.

I have the luxury of a home distiller that provides drinking water for our home that also makes a convenient and inexpensive source of distilled water for my use in my pretend science work.

You are a patient fellow to wait for settling, but as you say, it is likely the safest route.

Thanks

Rod

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Re: Doing Diatoms

#393 Post by rnabholz » Wed Dec 07, 2016 2:16 pm

The sample is ready for cleaning. I transferred it to a 250ml flask, bigger is always better in my experience, sometimes the reactions can be a bit unpredictable.
DSC_4763-468x832.JPG
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Adding the hydrogen peroxide, the immediate reaction is pretty calm which usually means that the sample is relatively clean. I placed the flask on the hot plate set around 175F and let it cook for about 2 hours.

Removing it from the heat, and let it cool for about a half hour. I place the flask on a catch tray (just in case) and add the equivalent of about half a baby aspirin of Potassium Dichromate to the flask. The sample instantly changes color to dark purple, and reaction begins to ramp up.

Thirty seconds later, it is in a vigorous boil and steam is shooting out of the top of the flask. The material is comfortably contained though, and 2 minutes later, the excitement is over and the orange color that signals the completed reaction appears.
DSC_4765-444x789.JPG
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I use a pipette to gather the sample from the flask and place it in two centrifuge tubes, and to them off with water.

Spinning them for 10 minutes at around 100rpm produces this result. Notice the difference in volume now that the organics have been removed.
DSC_4767-444x789.JPG
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I consolidate the material into one tube and do five rinse cycles until the orange color is removed.

I will take a look at the cleaned sample under the scope, and if all looks good, we will be making slides soon.

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Re: Doing Diatoms

#394 Post by zzffnn » Wed Dec 07, 2016 8:42 pm

Cold acid soak did significantly improve frustule cleanness to my eyes and I think it is definitely worth the effort. I apologize as I did not keep a comparison sample, so you would have to take my words for it.

This is the what the cleaned frustules in a water strew looked like, under 80x total magnification and 8x NA 0.2 achro objective. I used darkfield, as it would brutally reveal any none diatom stuff:
darkfield2.jpg
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It looks cleaner under oblique.
oblique2.jpg
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What I did was:
1) added 4 parts of 31.45% muriatic acid to 1 part of wet sample, covered up, soaked for 24 hours at outside temperature (around 50-60 F at the time);

2) added about more than enough amount (about 1 part, till color is deep purple) of saturated potassium permanganate solution to 1), covered up, soaked for 17 hours at outside temperature (around 55-65 F at the time);

3) took off lid and let stand for one hour, to release chlorine gas that had built up;

4) added more than sufficient amount of 3% hydrogen peroxide, stopped till bubbling had stopped and reaction color change had stopped (reaction color changed from dark grey to chlorinated water color / greenish yellow);

5) let stand for one hour, then took precipitates and washed with distilled water;

6) made a water strew slide, examined under scope with darkfield and oblique at 8x NA 0.20 (total magnification was 80x).

Please note:

I did not have 35% hydrogen peroxide, so I used chlorine as bleaching agent. Step 2 produced (toxic) chlorine gas as bleaching agent (without changing acidity of the reaction - if you use household bleach, reaction PH will turn basic and diatom frustules may be digested).

Based on what I saw under the scope, I think using 35% hydrogen peroxide at step 2) and some degree of bubbling would work better – some frustules are not fully exposed. 35% hydrogen peroxide at step 4) as well.

I apologize for the poor image quality - the 8X achro objective is not my usual photography objective and I did not have a good oblique set-up for it.

31.45% muriatic acid is highly corrosive and still produce toxic fumes. Chlorine gas is toxic as well. I worked on my balcony, wore water-proof gloves / long clothing / eyeglasses and held my breath while handling steps 1) – 5).

Boiling point of muriatic acid, based on Wikipedia, is a bit lower than water. So I am not sure it is a good idea to boil muriatic acid solution - you would probably vaporize away more acid than water (you would reduce acid concentration, when you boil it). On the other hand, reaction speed should increase as temperature rises, so those two factors will play against each other, the end result will depend on which factor wins over.

Take a look at how corrosive muriatic acid is:

1. Food digested in 33% HCL (muriatic acid) for 3 hrs
https://www.youtube.com/watch?v=D3AsFNX_CGs

2. 23% HCL took 2 days to dissolve chicken wing
https://www.youtube.com/watch?v=6tm_GNShWvo

3. chicken leg left overnight in HCL, H2SO4 and HF
https://www.youtube.com/watch?v=oipksRhISfM

Kurt,
You can take one gallon of 31.45% muriatic acid from me, when we are meeting in person next time, if you want. Home Depot had to sell two gallon-sized bottles at one time, I only need one.

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Re: Doing Diatoms

#395 Post by rnabholz » Thu Dec 08, 2016 12:51 am

So if I wasn't intimidated enough, you had to throw in the chicken leg videos.....Sheesh! ;^)

It seems to be a bit safer protocol than hot acid, and so that is good, and the results you report are good as well.

I am afraid the opportunity for me to give this a try may have sailed for the season. Daily high temps of 23F here today with the forecast dropping into single digits early next week means my outdoor lab is closed for business for a bit.

I am curious about the strength of acid necessary needed to be effective. Judging by the video, the 20% did work although taking longer. But we are not dealing with flesh and bone here. just some chloroplast and vegetable matter, which one would think would not put up near as big a fight. So if I could trade a little time for a less dangerous, lower fuming acid, that might be a good trade for me, the guy who has no formal training in handling this sort of thing.

Nice job and great report Fan. Thanks

Rod

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Re: Doing Diatoms

#396 Post by rnabholz » Thu Dec 08, 2016 1:16 am

Here is the first look at the cleaned diatoms in a wet mount. This is denser than I hope to have in the finished slides, but this was just a scouting mission.

Seen in Phase Contrast



On to slide making!!

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Re: Doing Diatoms

#397 Post by zzffnn » Thu Dec 08, 2016 1:30 am

Rod,

Cold acid, even in high concentration, should be OK to handle, as long as you don't get it on your skin or into your lungs.

The results of those videos are not directly comparable, since the end results were not quantified (e.g., how many grams of meat is fully digested).

I read from material safety data sheet that muriatic acid is considered as a significant corrosive agent, when concentration is over 25%. I think FINAL concentration close to 25% would work - my final concentration was at around 24%, for example.

Up to 32%, risk of toxic fume does not increase significantly, while effectiveness does increase quite a bit.

Pond for pond, vegetable matter may actually stay longer than meat, in muriatic acid. This is because plant fiber (polysaccharides) is harder to digest than meat (proteins and lipids) for HCL. But a soak of 1-2 days should be enough for anything.

It is possible to do acid soak indoor, as long as you can trap the escaping fume with baking soda water. And make sure that your children/family do not touch it.

You can cover the acid container with aluminum foil, then invert a huge glass container over the acid container, fully covering the acid container, then fully submerge the rim of the huge container with baking soda water. Regular water is fine too, if you are too lazy to make baking soda water. The same method can contain and neutralize chlorine gas too.

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Re: Doing Diatoms

#398 Post by zzffnn » Thu Dec 08, 2016 1:41 am

rnabholz wrote:Here is the first look at the cleaned diatoms in a wet mount. This is denser than I hope to have in the finished slides, but this was just a scouting mission.

Seen in Phase Contrast



On to slide making!!
Interesting. Diatom forms in my latest Armand Bayou sample is not too different than this lake sample, except that mine has a few Pleurosira laevis here and there. I just look at my cleaned sample carefully. You don't have to clean that Armand Bayou sample, by the way, unless you really like it. Concentration in my sample seems less.

What was the total magnification of your phase video?

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Re: Doing Diatoms

#399 Post by rnabholz » Thu Dec 08, 2016 2:12 am

zz,

As always thanks for the information. I don't see myself taking any chances with indoor processing with acid - I don't have the confidence that I wouldn't foul it up, and the penalty that might involve harm to my family is beyond the pale for me. Maybe in spring when I can get outdoors again.

So far I have made out ok for my purposes with relatively less dangerous chemicals, and have at least some comfort level with them. I think I will play this hand for a while.

The video was afocal through the 20x objective and 10x eyepieces - no zoom on the camera.

Rod

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Re: Doing Diatoms

#400 Post by zzffnn » Thu Dec 08, 2016 2:21 am

Sounds good, Rod. I understand.

I forgot to say that the lake sample has some more pennate forms than the latest Armand Bayou sample too.

The acid test was ran on left-over wash from the Armand Bayou sample. You may have more concentration there, than what was in my photos. Form diversity may be similar.

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Re: Doing Diatoms

#401 Post by rnabholz » Thu Dec 08, 2016 3:11 am

A quickie image from the first Leisure Lake Sample slide.
Rhopolodia 80µm
Rhopolodia 80µm
Rhopolodia 3048.JPG (48.26 KiB) Viewed 7056 times
More to come.

***********************************************
More Leisure Lake Diatoms can be seen here:


viewtopic.php?f=6&t=4301

viewtopic.php?f=6&t=4311

***********************************************
Last edited by rnabholz on Tue Dec 20, 2016 9:55 pm, edited 2 times in total.

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Re: Doing Diatoms

#402 Post by Dale » Thu Dec 08, 2016 3:39 am

Will we have to wait for Santa to get a review on the tach? I read about it. Many bad reviews, but
many reflected an inability to read the directions! One user took out the circuit board and mounted
it inside his centrifuge. It does draw 300 microamps 24/7.
Thanks for the tip about using 250 ML flasks, was all set to buy 125's.
Until I finish wiring a new computer I will have no desk space to work on the Ocean Shores sand. If
I wait too long, well I guess the cats will like it!
If I just wait you guys answer all my questions before I have to ask them. Now if the carpenters would
just finish my house........
Dale
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Re: Doing Diatoms

#403 Post by lorez » Thu Dec 08, 2016 3:55 am

I'm glad to see the sample is productive.

lorez

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Re: Doing Diatoms

#404 Post by rnabholz » Thu Dec 08, 2016 4:59 am

lorez wrote:I'm glad to see the sample is productive.

lorez
Thanks for making the effort to collect and mail it to me. It looks to have some good stuff in it.

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Re: Doing Diatoms

#405 Post by rnabholz » Thu Dec 08, 2016 5:09 am

Dale wrote:Will we have to wait for Santa to get a review on the tach? I read about it. Many bad reviews, but
many reflected an inability to read the directions! One user took out the circuit board and mounted
it inside his centrifuge. It does draw 300 microamps 24/7.
Thanks for the tip about using 250 ML flasks, was all set to buy 125's.
Until I finish wiring a new computer I will have no desk space to work on the Ocean Shores sand. If
I wait too long, well I guess the cats will like it!
If I just wait you guys answer all my questions before I have to ask them. Now if the carpenters would
just finish my house........
Dale

The kids are always asking for my Christmas list, so I put the tach on it, so yes we may have to wait for Santa if you are looking to me for a review.

Really, I see using it to measure speed once to get speeds at various settings on the analog dial, and only checking it again after that from time to time. I can pull the battery if it draws when not on.

Depending on the size of your samples, you might want to have a 500ml beaker or two around as well. Bigger is better.

Good luck with the carpenters.

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Re: Doing Diatoms

#406 Post by Charles » Thu Dec 08, 2016 12:39 pm

Looks like a wonderful Leisure Lake sample Rod!

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Re: Doing Diatoms

#407 Post by Charles » Thu Dec 08, 2016 12:49 pm

zzffnn wrote:Rod,

Cold acid, even in high concentration, should be OK to handle, as long as you don't get it on your skin or into your lungs.

The results of those videos are not directly comparable, since the end results were not quantified (e.g., how many grams of meat is fully digested).

I read from material safety data sheet that muriatic acid is considered as a significant corrosive agent, when concentration is over 25%. I think FINAL concentration close to 25% would work - my final concentration was at around 24%, for example.

Up to 32%, risk of toxic fume does not increase significantly, while effectiveness does increase quite a bit.

Pond for pond, vegetable matter may actually stay longer than meat, in muriatic acid. This is because plant fiber (polysaccharides) is harder to digest than meat (proteins and lipids) for HCL. But a soak of 1-2 days should be enough for anything.

It is possible to do acid soak indoor, as long as you can trap the escaping fume with baking soda water. And make sure that your children/family do not touch it.

You can cover the acid container with aluminum foil, then invert a huge glass container over the acid container, fully covering the acid container, then fully submerge the rim of the huge container with baking soda water. Regular water is fine too, if you are too lazy to make baking soda water. The same method can contain and neutralize chlorine gas too.
It is very good to be cautious when using acids, but I fear you can get overly cautious too. HCL is what digests your food in the stomach. Have you seen how Coke can even dissolve items...even teeth left to soak in it? I use HCL regularly in my swimming pool to maintain pH and I use it to soak/clean the chlorine generator for my pool. Of course you don't want to ingest HCL or get it on you, but it is safe if handled properly. I have two heater/stirrers, one which I keep in the garage. When I cook the samples in HCL, I just take it outside and let it cook for a couple hours. I also use a water bath to bring the sample being cooked in HCL up to boiling. I check on it about every 20-30 minutes and top off with more HCL if too much is evaporated off.

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Re: Doing Diatoms

#408 Post by zzffnn » Thu Dec 08, 2016 1:36 pm

Charles,

I am actually less cautious, when I am doing it myself. I would do it in my indoor garage like how I mentioned it (with water), if my children don't touch it. But Rod has his reason to be more safe than sorry, I guess.

Sure, toxicity depends on how much, where and how long. Even salt and water can be toxic, even one takes in too much. For HCL, I just hate the smell, it is not like the short exposure can really damage my lungs, when I handle it outside - I did not have to hold my breath.

You know my wife does not let our children drink coke often, precisely because it is not good for their teeth. But I am more liberal on that. I even prefer steak with a little pink/blood inside, even though I know very well that there could be E. coli there - and I used to be a bacteriologist :shock: After all, what fun is life, if one lives according to textbooks, right :mrgreen:

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Re: Doing Diatoms

#409 Post by rnabholz » Thu Dec 08, 2016 3:39 pm

Thanks Charles and zz

I readily admit that I am probably over cautious regarding the acid. I am relatively sure that at some point in the near future you will read about me giving it a try. I just need to get more comfortable with the processes, procedures and protections.

Just using the hydrogen peroxide and potassium dichromate I have more than one boil over, and while a bother, the cleanup is simple and pretty safe. I was never that concerned. The same event with acid would be a different matter, and so I want to be as educated as possible to avoid dumb mistakes.

Please keep discussing your processes and experiments using the acid protocols. The more I (and I suspect others) read about it the safer we will be.

Thanks

Rod

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Re: Doing Diatoms

#410 Post by zzffnn » Thu Dec 08, 2016 4:04 pm

Rod,

Acid spill can be firstly neutralize with baking soda, then simple wipe off (once added with enough baking soda, acid turns into salt and carbon dioxide that are much less harmful than dichromate).

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Re: Doing Diatoms

#411 Post by zzffnn » Sun Dec 18, 2016 6:30 pm

Rod and Kurt,

What paint brush do you use for ringing slides?

A friend of mine is making some wooden birds and could use a super fine stiff brush. There is what his end product looks like: https://www.flickr.com/photos/bushman_k ... hotostream

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Re: Doing Diatoms

#412 Post by rnabholz » Mon Dec 19, 2016 3:18 am

Mine is from Hobby Lobby, and is a Master's Touch Liner #20.

I think Kurt uses a finer brush.

Rod
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Re: Doing Diatoms

#413 Post by KurtM » Tue Dec 20, 2016 4:53 am

I'm away from home right now so can't tell you the brushes I use for slide ringing. Anyone shopping for brushes should go the nearest Hobby Lobby for a wide selection to ponder over; and if in Houston, Texas Art Supply is an even richer trove.

Brushes are a notoriously personal (preference) thing - what works best for one guy is likely to be very different from what works best for the next.
Cheers,
Kurt Maurer
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email: ngc704(at)aol(dot)com
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Re: Doing Diatoms

#414 Post by rnabholz » Sun Jan 01, 2017 8:32 pm

The age old problem of what to give a Diatomist for Christmas, haven't we all wrestled with that one?

I thought I would share what my 20 year old daughter came up with in response to that dilemma.
Diatom Christmas Present 2016
Diatom Christmas Present 2016
Diatom Christmas 2016.jpg (146.62 KiB) Viewed 6854 times
She took the time to go out and collect 6 samples from different bodies of water near where she attends college. She told me that she watched a couple of videos on You Tube to learn how and she was off.

What a great kid!

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Re: Doing Diatoms

#415 Post by Charles » Sun Jan 01, 2017 11:16 pm

What an awesome and resourceful daughter you have Rod!

What a wonderful present!

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Re: Doing Diatoms

#416 Post by Dale » Sun Jan 01, 2017 11:55 pm

Was there a tachometer in your stocking?
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Re: Doing Diatoms

#417 Post by KurtM » Mon Jan 02, 2017 12:58 am

Charles wrote:What an awesome and resourceful daughter you have Rod!

What a wonderful present!
Yes sir, that's the coolest thing I've seen in long time - heck, she brightened up a few days with that one! 8-)
Cheers,
Kurt Maurer
League City, Texas
email: ngc704(at)aol(dot)com
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Re: Doing Diatoms

#418 Post by rnabholz » Mon Jan 02, 2017 1:02 am

Charles wrote:What an awesome and resourceful daughter you have Rod!

What a wonderful present!
Thank you Charles - she really is a great young woman. Full time school, part time job, and she still found time to go out and do this for me. Really proud of her.

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Re: Doing Diatoms

#419 Post by rnabholz » Mon Jan 02, 2017 1:10 am

Dale wrote:Was there a tachometer in your stocking?
Why yes there was!

It seems to be a perfectly good tool.

Here it is with its carrying case.
Tach.jpg
Tach.jpg (137.78 KiB) Viewed 6845 times
Inside the case is a substantial length of reflective tape. The tape needs to be applied to whatever mechanism that you want to measure. A very small square is all that is necessary. Seen here on one of the arms of my centrifuge.
Tach Tape.jpg
Tach Tape.jpg (138.04 KiB) Viewed 6845 times
The tach emits a red light, not sure if it is anything more than a red LED.
Tach Light.jpg
Tach Light.jpg (81.59 KiB) Viewed 6845 times
To take a measurement, you just center the light over the tape and press the Test button, turning on the light. The readout is immediate.
Tach test.jpg
Tach test.jpg (112.32 KiB) Viewed 6845 times
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. What I can say is that it is consistent and repeatable and so at least to that degree is useful, at least for my purposes. If I had my old turntable maybe I could run a test.... ;^)

At less than $20 shipped, I think it is a good value.
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Re: Doing Diatoms

#420 Post by rnabholz » Mon Jan 02, 2017 1:12 am

KurtM wrote:
Charles wrote:What an awesome and resourceful daughter you have Rod!

What a wonderful present!
Yes sir, that's the coolest thing I've seen in long time - heck, she brightened up a few days with that one! 8-)
Thanks Kurt,

I was really touched by it. I am a very lucky fellow.

Rod

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