My diatom adventures by Bill Dailey

[bill2penn]

As many of you know I no longer produce diatom mounting media Pleurax and Zrax since I have retired from the University of Pennsylvania Department of Chemistry. But I thought I would describe the history and procedures I developed since it is hard to find good mounting media. I have already provided my procedures to several people throughout the world, but I would like to make sure that my work and its history is not lost when I someday go to the great beyond. Part of my impetus for this was the passing of Klaus Kemp a year ago. Klaus had intended to have his formula for his "secret" liquid diatom adhesive be released posthumously by his wife Sheila. However, Sheila tragically passed before Klaus so his written formula was not released. However, I believe I have now reproduced Klaus' adhesive formula and will describe it later in this series.

What I ask, however, is that if anyone uses my Zrax procedure, that they not call their product Zrax. I spent many, many hours doing quality control to make sure my Zrax was good quality, and I am unable to do quality control of other people's material. You can call your product "Dailey's Zrax formula", or Improved Naphrax (which it is!), or something similar. I would like to keep the simple term "Zrax" for material that I personally prepared. Ok, now on to history to start the story...

I started my journey into diatoms in 2001 when I was just starting out in microscopy. I wanted something to view under the scope and diatoms seemed like a good thing. I found Klaus Kemp's website and ordered some of his prepared slides. I was AMAZED at what he sent. You can see a version of Klaus' website from the time by using the Wayback Machine here:

https://web.archive.org/web/20010420174855/http://www.diatoms.co.uk/

I asked Klaus if he could teach me how to do this, and he agreed. Stephen Nagy was his first student and had been working with Klaus for several years. Starting in January of 2002, Klaus, Stephen and I worked together to improve procedures for diatom cleaning and diatom mounting. One of the areas which needed a lot of work was mounting media. In 2001 there were not many options for high refractive index mounting media, and you had to make your own. There were the patents for Naphrax, recipes for Pleurax, and some leftover bottles of Hyrax. Since I am an Organic Chemist, it was logical that I would be the one to work on making the mounting media. I read everything I could and attempted to reproduce the patents describing Naphrax. You can see the patents here:

https://patents.google.com/patent/US2312329A/en?oq=us2312329

https://patents.google.com/patent/US2502446A/en?oq=us2502446

The chemical procedure descriptions in the two patents are just terrible. That's why most of the Naphrax produced is so poor. I was unable to get anything useful when I tried the published procedures for Naphrax, so I went on to try to make a batch of Pleurax which seemed much more straightforward reaction procedure. However, I found that when using very pure phenol and sublimed sulfur with dry sodium carbonate it gave NO REACTION! As is sometimes the case in synthetic chemistry, some water is actually required for a reaction to proceed. So I deliberately added some water and it worked! I found that removal of excess phenol by distillation increase the refractive index of the material. My procedure is as follows:

A 1 liter round bottom flask is filled with 500 grams of 99+% pure phenol, 220 grams of sublimed sulfur, and 0.25 grams of sodium carbonate dissolved as much as possible in 0.25g water. An air-cooled condenser is attached and the top of the condenser leads to trap and then to an oil bubbler. The outlet from the oil bubbler leads to a on gallon aqueous solution of ca. 10% sodium hydroxide to absorb the hydrogen sulfide. The mixture is slowly heated to a gentle reflux using an oil bath and is heated for 24 hours. While the orange solution is still hot, the condenser is fitted for distillation under aspirator pressure and the excess phenol is distilled from the mixture up to an oil bath temperature of 160 C. The mixture in the pot is cooled slightly, diluted with 1 liter of acetone and filtered through 0,70 micron glass fiber filters (Milipore AP40) under vacuum. [Edit: I originally had written the wrong size of filter as 100 microns.] Several changes of filter may be required to achieve a fast enough flow. The filtered solution is evaporated on a hot plate in a large beaker and the Pleurax is ready to dissolve in the solvent of choice.

This procedure works well and reproducibly. The resulting product acts as a very good mounting material for strews but selected slides can't be done with it since the alcohol solvent will dissolve the gelatin, shellac, or tragacanth gum of the adhesives. At least that's what everyone believed at the time. I'm very stubborn so if someone says something can't be done, I have to try it to believe it. Well, it's true that the gelatin or tragacanth gum-based adhesives don't work with Pleurax, BUT THE MEAKIN DEXTRIN/GLYCERINE ADHESIVE DOES WORK!!!!  I found that I can make selected slides using the Meakin dextrin/glycerine wet adhesive. Klaus also found it to work at least on small arrangements. To prepare the Meakin adhesive I use 1 part dextrin (British Gum), 1 part water, and 3 parts glycerine. (Meakin used 1 part dextrin, 0.5 part water, 3 parts glycerine. However, I could not get all the dextrin dissolved in 0.5 part water) Warm the solution until dissolved, then either centrifuge on high for a day, or let it sit upright undisturbed for many weeks. The clear top layer is what you want. It's important to use yellow (not white) dextrin. If your dextrin doesn't dissolve in water, you don't have the correct dextrin. To use this, put a tiny drop on the glass and rub it well with your finger, then use a lens tissue to make a final swipe to clear any impurities. When you breath on the glass and look at it under the scope you should see very small lines of liquid. Place diatoms on the glass and slide them into position in order to make sure they pass through some of the liquid. Once all the diatoms are in place, heat the glass to remove glycerine (I use a spirit lamp). Then mount in Pleurax. Be careful not to heat the slide too hot too quickly since isopropanol (or ethanol) evaporates quickly and the vigorous bubbling can dislodge the forms.

This procedure worked really well for me for about 6 months, but then I became EXTREMELY allergic to Pleurax. Even today I break out in poison-ivy type blisters that last for weeks if I just look at Pleurax. So I had to stop making and using Pleurax. I needed to come up with another high refractive index mounting medium. That will be the next part of this story.


Bill

[Rene]

Thanks Bill, looking forward to your next chapter!
I believe Michel Haak used a different adhesive for Pleurax, based on polyacrylamide. Do you have any experience with that?
It wasn't a stable mixture though, he made it every couple of days again. His mix doesn't leave swipe-patterns on the coverslip that show up in DIC or PC. I always found that a bit of a bummer in Klaus Kemp's slides.

Best wishes, René

[anne]

Dear Bill,
very exciting story!
Klaus showed me, when I visited him exactly this kind of use of his adhesive.
Looking forward for the next chapter of the story.
lg
Anne

[martin_hu]

Hello bill

thanks for sharing your information!
I look forward to hearing more.

Best regards

Martin