Doing Diatoms a Different Way

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exmarine
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Re: Doing Diatoms a Different Way

#31 Post by exmarine » Wed Dec 07, 2016 11:10 pm

Members I can only say THANK YOU ALL for a Board Index that should go down as 'How it should be carried out'. Once it all drops into place it is beneficial to all diatomists. Can't think of what else to say regarding the thread except its so good if someone took the time to condense it all into a protocol it will stand the test of time.
I congratulate all those who have contributed to this thread I found it outstanding. Thank you.
Thank you :shock:
Best regards
exmarine :x

uses Watson 'Service' 1950 compound.
uses Watson Stereo 1960 ish.

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Re: Doing Diatoms a Different Way

#32 Post by Charles » Thu Dec 08, 2016 12:34 pm

KurtM wrote:Charles, any words of wisdom on how best to apply just the right amount of adhesive to cover slip?
The way I'm doing it is to wash you hands really well, especially your index finger. Get a clean coverslip and using a toothpick, pick up some adhesive on the tip and touch it to the coverslip. It should be a very small amount and not a glob. Dip your clean index finger in IPA and let dry. Then rub the adhesive on the coverslip between thumb and index finger until the adhesive becomes tacky. A final straight firm stroke with the index finger straight across the surface of the coverslip and it should be ready for mounting your forms.

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Re: Doing Diatoms a Different Way

#33 Post by Charles » Thu Dec 08, 2016 12:35 pm

exmarine wrote:Members I can only say THANK YOU ALL for a Board Index that should go down as 'How it should be carried out'. Once it all drops into place it is beneficial to all diatomists. Can't think of what else to say regarding the thread except its so good if someone took the time to condense it all into a protocol it will stand the test of time.
I congratulate all those who have contributed to this thread I found it outstanding. Thank you.
Thank you for your comments. It is a good thing to share techniques and ideas.

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Re: Doing Diatoms a Different Way

#34 Post by KurtM » Fri Dec 09, 2016 1:28 am

A final straight firm stroke with the index finger straight across the surface of the coverslip and it should be ready for mounting your forms.
Well, I certainly wasn't expecting that! But okay, I can dig it. Tell me, though: once the final film is in place, how long do you get for working time, before it dries too hard to have any tack left? Dumb question, I know, just need to try it for myself and find out. Probably will tomorrow (be too cold to do anything outside, even here on the Texas gulf coast).

Maybe get busy and come up with a manipulator too.
Cheers,
Kurt Maurer
League City, Texas
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Re: Doing Diatoms a Different Way

#35 Post by Charles » Fri Dec 09, 2016 1:37 am

It depends on what adhesive you use. The gum tragacanth and IPA will last weeks. You can start a mounting session and then leave it for the next day and the next until you're ready to add the mountant. When you have the forms where you want them, just breath on the coverslip to let the adhesive set the forms. Once all the forms are set, warm it on a hot plate and then use your mountant. If you do not finish the coverslip with adhesive the same day, be sure to cover it so dust does not get on it. I use a petri dish to cover it but anything which will keep the dust out.

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Re: Doing Diatoms a Different Way

#36 Post by Charles » Fri Dec 09, 2016 11:51 pm

Charles wrote:
KurtM wrote:I want to know about the adhesive. I've fooled around with arranging some, but hit a wall when it comes to putting them down as desired, and getting them to stick. But even if I were to get an arrangement successfully placed, I still have no idea how I'd cure Pleurax without the bubbling undoing the arrangement.

So yeah, you have a bunch of us stirred up pretty well with your post, I'd say. 8-) :lol:
I use an adhesive which Klaus Kemp uses. It is made of 1/3 finely ground gum tragacanth dissolved in 1/3 distilled water and 1/3 IPA with few drops of glacial acetic acid added after.

They say the key to keep the forms from moving is proper adhesive and gentle heating.
Looking at my notes from 2012, it looks like I left a key ingredient out of the above adhesive...Glycerin.
The recipe for the adhesive is as follows.

This is best done with a heater with a magnetic stirrer and your mixing beaker should be in a larger beaker or container of water to prevent scorching.

-Boil 20ml of water (the amount can be 30ml or whatever you want as long as you use equal amounts of Glycerin and IPA. Believe me, 50-60ml of this will last you a lifetime) in a beaker with magnetic stirrer rod in place.
-Activate the stirrer and slowly add the Gum tragacanth until the mixture is the consistency of heavy syrup.
-Turn off the heat.
-Add 20ml of Glycerin (the stirrer should still be going)
-Add 20ml of IPA (90% and the stirrer should be still mixing)
-Add 3-4 drops of Glacial Acetic acid.
-Let all ingredients mix well.
-Filter the mixture into a clean storage container.

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Re: Doing Diatoms a Different Way

#37 Post by KurtM » Sat Dec 10, 2016 12:55 am

Not two days ago I bought a new hot plate. I've been shopping for the right deal for weeks. Part of that was agonizing over whether to get one with a stirrer or not. I finally decided on not, since I've never had a need for stirring before.

Now if you'll excuse me, I'm going to bash my forehead on the desk repeatedly for a while ... sheeeesh... :roll:

In other news, my capillary tubes arrived today, and I amused myself for a short while by attempting to pull needles. I couldn't seem to get any that were really really small, like I hit a wall at half a millimeter or so. And then my alcohol lamp refused to burn denatured alcohol any more and petered out on me. It has been "one of those days" around here, so I quit.

Now I think I'll take up drinking heavily... :roll:
Cheers,
Kurt Maurer
League City, Texas
email: ngc704(at)aol(dot)com
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Re: Doing Diatoms a Different Way

#38 Post by zzffnn » Sat Dec 10, 2016 12:57 am

Thanks for the note, Charles.

I wonder what glycerine does in the glue. I am guessing:
1) it has a high refractive index and mixes well with water and other solvents;

2) it retains even distribution of moist/water and keep texture smooth;

3) it has an slight antioxidant activity and works partially as preservative, though IPA and acetic acid may be main preservatives there.

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Re: Doing Diatoms a Different Way

#39 Post by Charles » Sat Dec 10, 2016 2:32 am

KurtM wrote:Not two days ago I bought a new hot plate. I've been shopping for the right deal for weeks. Part of that was agonizing over whether to get one with a stirrer or not. I finally decided on not, since I've never had a need for stirring before.

Now if you'll excuse me, I'm going to bash my forehead on the desk repeatedly for a while ... sheeeesh... :roll:

In other news, my capillary tubes arrived today, and I amused myself for a short while by attempting to pull needles. I couldn't seem to get any that were really really small, like I hit a wall at half a millimeter or so. And then my alcohol lamp refused to burn denatured alcohol any more and petered out on me. It has been "one of those days" around here, so I quit.

Now I think I'll take up drinking heavily... :roll:
You can always stir it by hand...fork or small wire whisk. You only need to do it right just one time. ;)

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Re: Doing Diatoms a Different Way

#40 Post by Charles » Tue Dec 13, 2016 1:18 pm

This last weekend, I went to different beaches to get some sand for lorez. Ended up going to the lower James River, where it empties into the Chesapeake Bay, Buckroe Beach on the North shore of the Chesapeake Bay, and Virginia Beach, on the Atlantic Ocean. As I was rinsing the sand for lorez's collection, I kept the rinse water to look for diatoms. Winter time and cold waters makes for scarce diatom populations but I did see some interesting forms in some of the samples. Although scarce, each location had it's own different types of forms. I will go back to those sites come Spring and Summer to see what the population looks like.

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Re: Doing Diatoms a Different Way

#41 Post by zzffnn » Tue Dec 13, 2016 1:39 pm

Charles,

I am glad you can still find diatoms in winter. I found very little in December, from Galveston/Houston TX area. I did find that even on the same site (and at the same time), different host plants of materials can have different diatoms.

I also read that water salinity and sunshine are the main determinating factors for diatom population.

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Re: Doing Diatoms a Different Way

#42 Post by Charles » Thu Dec 15, 2016 12:46 pm

After processing the samples, I found a lot more diatoms than I thought was there. I did have to process about 150 ml of sand to get it, but it seemed to be well worth it.

As mentioned above, I took three location sand samples, Lower James River, where it meets the Chesapeake Bay, North shore of the middle of the Chesapeake Bay and the Southern part of Virginia Beach on the Atlantic.

The James River samples was taken near the shore on a small sandbar at low tide. Took samples from the tip of the sandbar and both sides near the shoreline.

The Chesapeake bay sample was taken at Buckroe Beach again at low tide, on both sides of a jettie and brushings from some oysters attached to the jettie rocks.

The VA Beach sample was taken at low tide, near a jettie and some from the upper tide line.

I processed each by immersing about 150 ml of sand in tap water, swirling it around and pouring the water into a 150ml flask and repeating about three times or until the water looked clear.

I let the samples settle for 2 hours and then siphoned off the top water and then poured the bottom portion with sample into a 15 ml centrifuge test tube, but I did not centrifuge it. I just wanted that type of tube because of the pointed bottom tip to condense my sample.

After I let it settle into the tip of the centrifuge tube, I siphoned off the top water and added 30% H2O2 to within 1 inch of the top of the tube and gently boiled it for 1.5-2 hours by placing the tube in a 150 ml flask with water.

Then I added Dichro, a few grains at a time until the reaction stopped and it turned yellow.

After settling, I siphoned off the H2O2 and Dichro off and added 30% HCL to within 1 inch from the top of the tube and boiled for 1.5-2 hours again in a 150ml flask fill with water, on a second hot plate just outside the garage.

After cooling, I siphoned off the HCL and rinsed three times with tap water, letting the sample settle well between washes. Then rinsed three times with distilled water, again letting the sample settle well between washes.

So, no straining, no centrifuging, just taking the time to settle. What I got in return were large unbroken diatoms, with some which were still together with the top, bottom with girdle still attached. There were still some broken forms especially the larger forms but if you can take the time to clean the sample gently, you will be rewarded with some nice samples. I also had a lot of sand particles but for my purpose of strews to retrieve individual forms for mounting later, it was not a problem. I just diluted the sample well so I can pick out the diatoms I want.

The sample includes Odontella, Terpsinoë, Auliscus, Actinoptychus, gyrosigma, a few Triceratium and even a Hydrosera. There were also a variety of various other centrics and pennates.

Pictures of the samples under the Stereo scope to follow.

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Re: Doing Diatoms a Different Way

#43 Post by zzffnn » Thu Dec 15, 2016 12:57 pm

Nice work, Charles. Thank you for sharing.

Do you have a photo of the James River sandbar? I am just curious how it looks like.

I usually collect bear jetties too.

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Re: Doing Diatoms a Different Way

#44 Post by Charles » Thu Dec 15, 2016 1:17 pm

I did not take of picture of the site but here is a Google Maps photo with GPS coordinates, showing the area on the James River.
JamesRiver.jpg
JamesRiver.jpg (183.65 KiB) Viewed 7203 times

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Re: Doing Diatoms a Different Way

#45 Post by rnabholz » Thu Dec 15, 2016 2:38 pm

Excellent Charles!

My experience with the late fall collections was also good. Maybe the season is a bit longer than we think?

But this morning it is -9F, so any collection effort would start with an axe to get through the ice....

And the kinder/gentler approach to handling is also food for thought.

Tell me about your siphon technique/equipment. You certainly aren't sucking on a hose filled with acid?

Thanks.

Rid

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Re: Doing Diatoms a Different Way

#46 Post by Charles » Thu Dec 15, 2016 3:06 pm

rnabholz wrote: Tell me about your siphon technique/equipment. You certainly aren't sucking on a hose filled with acid?

Thanks.

Rid
I use disposable plastic pipettes with the bulb on the end. Like these:
http://www.ebay.com/itm/Topwel-2ml-5-3- ... SwImRYUaPi

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Re: Doing Diatoms a Different Way

#47 Post by rnabholz » Thu Dec 15, 2016 3:16 pm

Charles wrote:
I use disposable plastic pipettes with the bulb on the end. Like these:
http://www.ebay.com/itm/Topwel-2ml-5-3- ... SwImRYUaPi
Ok, I do the same. You used the term siphon, and my mind jumped to hose and suction.

Thanks.

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Re: Doing Diatoms a Different Way

#48 Post by zzffnn » Thu Dec 15, 2016 3:21 pm

Rod,
My much less elegant way is to use a turkey baster.

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Re: Doing Diatoms a Different Way

#49 Post by KurtM » Thu Dec 15, 2016 3:33 pm

Charles wrote:The sample includes Odontella, Terpsinoë, Auliscus, Actinoptychus, gyrosigma, a few Triceratium and even a Hydrosera.
Nicely done sir - you really add a lot to the discussion of diatom cleaning for the amateur, thank you! Two questions from here:

1. What sort of references do you use for ID?

2. My understanding was that HCI is necessarily a pre-treatment, used to eliminate calcites, and must be administered prior to any further processing for some reason I can't recall. You use it after H202/Dichro, why?
Cheers,
Kurt Maurer
League City, Texas
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Re: Doing Diatoms a Different Way

#50 Post by rnabholz » Thu Dec 15, 2016 4:03 pm

zzffnn wrote:Rod,
My much less elegant way is to use a turkey baster.
I have one of those too...

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Re: Doing Diatoms a Different Way

#51 Post by Charles » Thu Dec 15, 2016 5:30 pm

zzffnn wrote:Rod,
My much less elegant way is to use a turkey baster.
It's kind of hard trying to get a turkey baster down a centrifuge tube. :shock:

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Re: Doing Diatoms a Different Way

#52 Post by Charles » Thu Dec 15, 2016 5:36 pm

KurtM wrote:
Charles wrote:The sample includes Odontella, Terpsinoë, Auliscus, Actinoptychus, gyrosigma, a few Triceratium and even a Hydrosera.
Nicely done sir - you really add a lot to the discussion of diatom cleaning for the amateur, thank you! Two questions from here:

1. What sort of references do you use for ID?

2. My understanding was that HCI is necessarily a pre-treatment, used to eliminate calcites, and must be administered prior to any further processing for some reason I can't recall. You use it after H202/Dichro, why?
1- I have to use a variety of different sites to get IDs. WoRMsS is one:
http://www.marinespecies.org/photogalle ... &pic=16726

2- I got in the habit of using the H2O2/Dichro first in case there are foramin in the marine samples, since HCL will dissolve those. I don't know that one way is better than the other. I can also get most of the cleaning with the H2O2 inside and do the HCL outside last.

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Re: Doing Diatoms a Different Way

#53 Post by Charles » Thu Dec 22, 2016 12:45 pm

A sample of some of the different Virginia Beach diatoms taken with USB camera on B&L Stereoscope. Crappy pictures but, you can see some variety. Many of the forms are whole forms...top, bottom and girdle.
Coscinodiscus, whole and single forms, taken at 75X total magnification:
Large Coscinodiscus Centrics 75X.jpg
Large Coscinodiscus Centrics 75X.jpg (458.84 KiB) Viewed 7143 times
Odontella taken at 105X total magnification. You can see the whole form on the left:
Odontella 105X.jpg
Odontella 105X.jpg (511.86 KiB) Viewed 7143 times
Triceratium:
Triceratium.jpg
Triceratium.jpg (374.93 KiB) Viewed 7143 times
Actinoptychus, whole and single forms:
Actinoptychus Centrics.jpg
Actinoptychus Centrics.jpg (494.02 KiB) Viewed 7143 times
And UFOs...Unidentifed Forms and Objects...probably mostly spicules:
UFOs 105X.jpg
UFOs 105X.jpg (420.47 KiB) Viewed 7143 times
Last edited by Charles on Thu Dec 22, 2016 1:16 pm, edited 1 time in total.

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Re: Doing Diatoms a Different Way

#54 Post by Charles » Thu Dec 22, 2016 1:07 pm

And these are from the lower James River/Upper Chesapeake Bay.
Haven't figured out if these are Auliscus or Pleurosira:
Large Auliscus Centrics 105X.jpg
Large Auliscus Centrics 105X.jpg (504.2 KiB) Viewed 7143 times
Some Coscinodidscus:
Large Coscinodiscus 105X.jpg
Large Coscinodiscus 105X.jpg (412.48 KiB) Viewed 7143 times
Terpsinoe single, whole forms and colonial forms:
Terpsinoë 105X.jpg
Terpsinoë 105X.jpg (420.37 KiB) Viewed 7143 times
Hydrosera single and whole forms...top, bottom and girdle:
Hydrosera 105X.jpg
Hydrosera 105X.jpg (304 KiB) Viewed 7143 times
Odontella:
Odontella.jpg
Odontella.jpg (415.17 KiB) Viewed 7143 times

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Re: Doing Diatoms a Different Way

#55 Post by rnabholz » Thu Dec 22, 2016 1:47 pm

Outstanding work Charles!

What a bunch of fascinating forms you have gathered there.

My vote would be for Pleurosira, but you would be smart not to put to much confidence in one of my IDs

May I ask how much time is represented in these sorted groups?

Great post.

Rod

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Re: Doing Diatoms a Different Way

#56 Post by zzffnn » Thu Dec 22, 2016 2:16 pm

Nice work, Charles.

I will vote for Pleurosira too.

I am surprised that Virginia is so productive in winter! To get a good amount of Coscinodiscus in Galveston, for example, I have to wait for spring 2017 (few were found in my November hunt, even though I saw a few last spring). Our same lacks variety too.

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Re: Doing Diatoms a Different Way

#57 Post by billbillt » Thu Dec 22, 2016 3:51 pm

I enjoyed these!.. Very pleasing to look at..

BillT

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Re: Doing Diatoms a Different Way

#58 Post by Charles » Thu Dec 22, 2016 4:55 pm

rnabholz wrote:Outstanding work Charles!

What a bunch of fascinating forms you have gathered there.

My vote would be for Pleurosira, but you would be smart not to put to much confidence in one of my IDs

May I ask how much time is represented in these sorted groups?

Great post.

Rod
Thank you Rod,
Yup, Pleurosira seems more like it.

So far, I have made 10 strew slides of each of the three sites I took samples from (30 total). I have a corresponding storage slide for each of the three sites (I try to keep them separate so I can see what forms each area has). Even when I process them, each sample from a site gets its own flask, centrifuge tube and pipette so I don't cross contaminate them. Picking the forms from each slide takes about an hour or more. Recently I have only been picking the largest or unusual forms since there are so many small ones. I scan each strew slide from top to bottom moving the right and then go over them moving from the left. It's surprising how many forms I miss going just in one direction. When I discover an unusual form, like I did with the Hydrosera, I will go over the other completed strew slides again to hunt for these as my eyes will seek out a particular shape in girdle view as well as top and bottom view. The diatoms you see in the above is only about 10-15% of the total forms on the storage slides, as there are wide range and number of pennates and other centrics.
Here are some Pleurosigma:
GyroSigma 105X.jpg
GyroSigma 105X.jpg (469.54 KiB) Viewed 7116 times
Last edited by Charles on Thu Dec 29, 2016 1:13 pm, edited 2 times in total.

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Re: Doing Diatoms a Different Way

#59 Post by Charles » Thu Dec 22, 2016 5:16 pm

Thank you Z and Bill!

I was pleasantly surprised with the variety for a late fall collection. I did have to process about 150ml of sand to see it though. Just looking at the sample under a scope before processing showed very sparse coverage, but the processing seemed to condense them all.

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Re: Doing Diatoms a Different Way

#60 Post by Charles » Sat Dec 24, 2016 6:22 pm

Rod sent me some of his diatom samples and I've started processing them.

First is his Lake Michigan samples as noted here:
viewtopic.php?f=6&t=3780

The first picture is a diluted strew slide which shows large chains of centrics identified as Ellerbeckia arenaria in a field of mainly Cocconeis. The long chains looked like broken drainage tubing on a sea floor. Also there was like a film, almost spiderweb like which clung to everything, which made picking clean forms very difficult. My next step is to boil in HCL to see I can remove the film.

All pictures taken on the B&L Sterozoom with USB eyepiece camera.

The Ellerbeckia on the strew:
Ellerbeckia arenaria Colonial.jpg
Ellerbeckia arenaria Colonial.jpg (84.52 KiB) Viewed 7094 times
This is what they look like on the storage slide:
Ellerbeckia arenaria Colonial on Storage2.jpg
Ellerbeckia arenaria Colonial on Storage2.jpg (47.2 KiB) Viewed 7094 times
Some other forms, Cymbella:
Cymbella2.jpg
Cymbella2.jpg (33.82 KiB) Viewed 7094 times
Some Surirella, I even found a solitary Surirella spiralis (not in the picture)
Surirella2.jpg
Surirella2.jpg (39.38 KiB) Viewed 7094 times
Even some Gyrosigma:
Gyrosigma2.jpg
Gyrosigma2.jpg (35.96 KiB) Viewed 7094 times
Thank you Rod for the samples!

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