MicrobeHunter.com Microscopy Forum

Hello all! I’ve been experimenting with cleaning diatoms with some methods i read during my quarantine induced web browsing. So far I used just household bleach and it seems to work well, I will see if i can prepare a larger sample to clean. My next step is to mount any diatoms i clean, however it seems hard to find any. Pleurax is currently unavailable from diatoms.nl. And i would prefer to avoid any medium with harmful solvents. Are there any diy solutions that are long term? I read the PS-CBO post and might dabble in that. Thanks for the help everyone!

Timemaster1212 wrote: ↑

Thu Jun 18, 2020 9:15 pm

Hello all! I’ve been experimenting with cleaning diatoms with some methods i read during my quarantine induced web browsing. So far I used just household bleach and it seems to work well, I will see if i can prepare a larger sample to clean. My next step is to mount any diatoms i clean, however it seems hard to find any. Pleurax is currently unavailable from diatoms.nl. And i would prefer to avoid any medium with harmful solvents. Are there any diy solutions that are long term? I read the PS-CBO post and might dabble in that. Thanks for the help everyone!

The PS-CBO is useful for about 3-4 months. Afterwards, components of the CBO crystallize out and ruin the slide.
The coverslip rims must be sealed. However, nail polish is not compatible with it. Gel nail polish, of the type that is cured with UV, works.

For permanent slides, Naphrax appears to be available from Brunel.UK. The solvent is toluene. It is harmful. So precaution is needed when using it.

Other than dissolving the naphrax in the toluene, do I need any special precautions when using it? IE using it outside? I saw it available in North America through this link: https://www.phycotech.com/Products/Prod ... xport-pack

If this is my current only option for long term mounting i am willing to take the precautions. I would like to hear about any experience people have with it in an amateur sense.

Hi,
I would suggest to contact Michel from Diatoms.nl and ask when Pleurax is available again. The cooking of Pleurax is not that difficult so he probably just has to find the time to cook a new batch. His website changed a lot in recent time so the Pleurax might just have been lost in a digital way. In use Pleurax is very nice for strew slides.

In the mean time you can make dry mounts. The high refractive index difference between air and diatom material gives a crist view. The only limitation is that you can't use the benefit of oil immersion objectives with numeric aperture above 1.

Bob

Timemaster1212 wrote: ↑

Thu Jun 18, 2020 10:41 pm

Other than dissolving the naphrax in the toluene, do I need any special precautions when using it? IE using it outside? I saw it available in North America through this link: https://www.phycotech.com/Products/Prod ... xport-pack

If this is my current only option for long term mounting i am willing to take the precautions. I would like to hear about any experience people have with it in an amateur sense.

Naphrax has been the mountant of choice for diatom research. In well-equipped labs, I believe they do it in a hood, especially when preparing batches of slides.
I never worked with Naphrax. Here is a protocol, cited from "An Introduction to the Microscopical Study of Diatoms" by R.B. MacLaughlin (p. 143):

8.5.8. Mounting in Naphrax This mountant is very intolerant of moisture. The diatom material must be thoroughly dry before applying it. The cover, with diatoms, should be dried for at least 15 minutes at 80°C., then rinsed off with three changes of xylene or toluene, after which the Naphrax is applied. The mechanical procedures in handling coverglass and microslide are the same as described previously for other mountants. After the Naphrax is applied to the cover it should be put back on the hotplate for at least an hour before being joined to the microslide. The preparation is then heated further to drive off the remaining solvent. If overheated, Naphrax will become darker in color.
(the properties of Naphrax are described in page 120 of that book).

Most or all high RI diatom mounting media contain a harmful solvent, which is being evaporated and released since mounting demands heating of the slide. This step of the process calls for precaution.

I can provide small quantities of the following for interested amateurs;

Pleurax (in alcohol)

Naphrax (in toluene)

Filtered Storax (in chloroform)


You need to PM me to enquire. Sending from the UK.

MicroBob wrote: ↑

Fri Jun 19, 2020 7:38 am

Hi,
I would suggest to contact Michel from Diatoms.nl and ask when Pleurax is available again. The cooking of Pleurax is not that difficult so he probably just has to find the time to cook a new batch. His website changed a lot in recent time so the Pleurax might just have been lost in a digital way. In use Pleurax is very nice for strew slides.

In the mean time you can make dry mounts. The high refractive index difference between air and diatom material gives a crist view. The only limitation is that you can't use the benefit of oil immersion objectives with numeric aperture above 1.

Bob

I sent an email asking this exact question and he is no longer shipping pleurax at this exact time so i guess we will just have to see what happens. And I never even thought of dry mounts, what is a good procedure for diatoms to dry mount?

I hope that the Pleurax from the Netherlands will become available again...

Dry mounts: I have done this only by accident by letting dry diatoms in salt water.
When you have cleaned diatoms you might try to slightly fix them to a slide by melting them into a thin layer of shellac. Then cover them with a cover slip that is held in place by a ring of UV or epoxi glue. This way they wont move to one side when the slide is held vertically.

Bob

Could the UV hardening resin be used as a mountant?

On a related note, i found a helpful article with my endeavors in diatoms http://www.microscopy-uk.org.uk/diatomi ... _Royal.pdf

While browsing the mountant chapter, i noticed that out of most, if not all, of the possible mountants, Storax seems the easiest and safest to produce in a home lab. Is there by chance anyone has attempted this lengthy process? It seems like an interesting summer project. I was considering this alternative if Pleurax remains unavailable for the time being.

Timemaster1212 wrote: ↑

Fri Jun 19, 2020 10:40 pm

Could the UV hardening resin be used as a mountant?

Hobbyst46 used NOA61, a UV glue. It works but it doesn't penetrate the frustules as dependably as Pleurax does.
Pleurax is comparatively easy to cook but you would absolutely need a laboratory fume cupboard. In recent times the potential durability of Pleurax slides has been seen as better as that of Naphrax slides.

Timemaster1212 wrote: ↑

Sun Jun 21, 2020 1:40 am

While browsing the mountant chapter, i noticed that out of most, if not all, of the possible mountants, Storax seems the easiest and safest to produce in a home lab. Is there by chance anyone has attempted this lengthy process? It seems like an interesting summer project. I was considering this alternative if Pleurax remains unavailable for the time being.

I never tried storax/styrax, but:

IMHO, trying to mount diatoms in styrax is worthwhile if you have the pure resin ready for use (according to that book chapter, styrax is NOT available). Even then, a solvent is needed (chloroform, toluene or xylene); logically, the solvent must be eliminated during mounting, so the slide must be heated. All in all, it appears to be neither easier nor safer than Naphrax or Pleurax.

If storax, the crude raw material, is your starting point - the situation is much worse. Workup of a practical quantity of raw material to isolate the pure styrax resin requires chloroform and/or toluene and/or xylene in quantities of the order of 0.1 liter (at least) and lengthy operations involving boiling. IMHO - don't even think of it, unless you work in a well-equipped lab and have some experience with such operations.

In fact, the time-consuming, non-modern styrax resin isolation from the storax raw material, as described by MacLaughlin, calls for the development of a better, faster, simpler, safer, reproducible process; since the storax plant is a source of pharmacy and medicine-related compounds, the literature possibly suggests some newer routes, or will soon reveal some.

How “unsafe” do you think this process would be? I am under the impressions it can be done safely at home with the right precautions. Then again i am usually one to look on the bright aide of issues haha. And correct me if i am wrong but would alcohol and acetone not be suitable to dissolve the storax to filter and spread it on the windowpanes?

Timemaster1212 wrote: ↑

Sun Jun 21, 2020 12:27 pm

How “unsafe” do you think this process would be?

Absolutely and totally unsafe ! chloroform is a suspected carcinogen, and is toxic from other aspects as well - read the MSDS.
It is very volatile, so vapors readily form, and, being heavier than air, could accumulate near the bench.

am under the impressions it can be done safely at home with the right precautions.

I would not, but do it at your own risk.

And correct me if i am wrong but would alcohol and acetone not be suitable to dissolve the storax to filter and spread it on the windowpanes?

1. I do not know. Chloroform is often (not always) a stronger solvent than those. Also, to separate and remove the unwanted components of storax, the solvent should be immiscible with water. Chlorofom is such, whereas alcohol and acetone mix well with water.
2. Alcohol and acetone (especially acetone) are highly flammable, the vapors are explosive at high concentration. So boiling such liquids at home calls for utmost precaution.

Ah thank you for the insight. Maybe some experiments with the cleaning process are in order for a safer method haha. Thank you for all your help!

I just got a vial of pleurax from Michel at diatoms.nl. the first week of July. It took about two weeks to arrive from the time of purchase. It cost me about $70 for 20ml. There was no charge or delay by customs. Michel is really pleasent to deal with.

Keith

photomicro wrote: ↑

Fri Jun 19, 2020 1:45 pm

I can provide small quantities of the following for interested amateurs;

Pleurax (in alcohol)

Naphrax (in toluene)

Filtered Storax (in chloroform)


You need to PM me to enquire. Sending from the UK.

I am an amateur diatomist and I desperately need a small supply of Pleurax for my last samples. I am studying high sltitude aquatic environments in Italy. please contact me at giobat99@gmail.com if you can help

I have tapped a big American styrax tree and it is very slowly producing resin. Should have a good crop in the spring I'll check back if I get enough to distribute

maurbatt wrote: ↑

Sat Oct 10, 2020 4:51 pm

photomicro wrote: ↑

Fri Jun 19, 2020 1:45 pm

I can provide small quantities of the following for interested amateurs;

Pleurax (in alcohol)

Naphrax (in toluene)

Filtered Storax (in chloroform)


You need to PM me to enquire. Sending from the UK.

I am an amateur diatomist and I desperately need a small supply of Pleurax for my last samples. I am studying high sltitude aquatic environments in Italy. please contact me at giobat99@gmail.com if you can help

Perhaps you could consult with Stefano Barone about a mounting medium. He produces really amazing and remarkable diatom stuff in Italy.

BramHuntingNematodes wrote: ↑

Sat Oct 10, 2020 4:57 pm

I have tapped a big American styrax tree and it is very slowly producing resin. Should have a good crop in the spring I'll check back if I get enough to distribute

Hi Bram,
I didn't have Styrax very bright on my radar. I would be interested to hear what you are doing and how you are attempting to make Styrax.
Bob

This is gonna sound ridiculous but I've done it with good result. There two absolutely non toxic mounting mediums that I have found.
1. Polyethylene glycol. RI of about 1 46. Dries perfectly clear.
Formula Buy Miralax or Clearlax. Mix two daily doses in 1/4 cup of warm water slowly stirring until completely dissolved. Let cool and use as mounting medium.

2. Poly vinyl acetate. RI about the same as above.
Source. Good old Elmer's Clear School Glue. Sounds dumb but slides do not yellow and last for years with proper care . a bottle If it is less than 3 dollars. Try it on something less important first.

With both of these substances slides dry completely in about 24 hours.

Good luck.
Greg

Hi Greg,
have you used these on diatoms?
Usually one tries to take a mountant that is at least 0,1 away from the ca. 1,45-1,5 refractive index of diatoms to make the clear diatom frustules visible with good contrast.

Bob