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PostPosted: Mon May 13, 2019 8:29 pm 
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Tried glycerin to slow down protozoa. Environmental disaster.
What wasn't killed outright, exploding or horribly deformed, looked like this one. (A few were slightly more mobile)
The survivors were flattened to the point that all internal functions are shut down. All trichocysts seem to have fired, or the alveoli are swollen.



Radazz

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PostPosted: Mon May 13, 2019 9:58 pm 
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When adding any of the viscous liquids to slow protozoa motion, better add very small quantities. Even a few percent are, in my experience, too much. Also, I think that methyl cellulose and similar polymers are better than glycerol, since their osmotic effect (for the same volume or weight) is much lower. There is a recipe by charlie g, cited by 75RR in a previous post from December 23, 2018. Search "cellulose" in the forum.

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PostPosted: Tue May 14, 2019 8:25 pm 
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I just had a chance to try out copper sulfate and copper acetate for, as the instructions say, "immobilizing fast-moving ciliates."

Results:
Copper sulfate (one drop per drop of pond water) - indeed all ciliates on the slide were completely immobilized.

Copper acetate (apx 1/4 drop per drop of pond water) - same result. Ciliates immobilized.

The water drop feels pretty dead with all the pond favorites immobilized, but I suppose if there is a need to have ciliates completely immobilized to study an internal structure or take a photo, this is a viable method.

On an unrelated note, I may have gotten a photo radar ticket today. Darn it. if so, there goes microscope/hobby money down the drain for penance of my crime. But there's hope 'til it arrives in the mail. :cry:


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PostPosted: Tue May 14, 2019 8:43 pm 
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Sauerkraut wrote:
I just had a chance to try out copper sulfate and copper acetate for, as the instructions say, "immobilizing fast-moving ciliates."

Results:
Copper sulfate (one drop per drop of pond water) - indeed all ciliates on the slide were completely immobilized.

Copper acetate (apx 1/4 drop per drop of pond water) - same result. Ciliates immobilized.


I should have mentioned that the solutions are from the Innovating Science stain kit purchased from AmScope. Copper(II) Sulfate 1% aqueous solution. Copper (II) Acetate 3% aqueous solution.


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PostPosted: Wed May 15, 2019 2:15 pm 
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Please try 1.5% methyl cellulose..50/50 mix of your specimen fluid and this viscosity tool. Please purchase the larger size rectangular cover slips so you never approach the wetmount cover slip borders with 'tight working distance' high mag objectives.

You can enjoy ciliates, large bacteria slowed down yet still patterning their usual body motions and ciliature wave propagations...a Parameciums 'fields of swaying ciliature' are beguiling..especially when the protist elects to change direction of body motion.

Enjoy live protists and use your oil-immersion objectives, with this 1.5% methyl cellulose viscosity tool. Charlie guevara


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Last edited by charlie g on Wed May 15, 2019 2:42 pm, edited 1 time in total.
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PostPosted: Wed May 15, 2019 2:42 pm 
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charlie g wrote:
Please try 1.5% methyl cellulose..50/50 mix of your specimen fluid and this viscosity tool. Please purchase the larger size rectangular cover slips so you never approach the wetmount cover slip borders with 'tight working distance' high mag objectives. --Charlie guevara


Thank you. Good to know. I only tried the other reagents because they unexpectedly came with a staining kit. Trial and error. Mostly error.


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PostPosted: Sat May 25, 2019 7:03 pm 
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Hi together,
we had our group meeting today and met in a restaurant afterwards with a couple of members. One topic was slowing down fas placton animals. One member reported a couple of methods he has applied with success:

-Osminumtetroxyd-fumes (Absolutely not recommended, highly toxic, he found a bottle in a hospital cupboard in Thailand and made a few tests)
-Cocaine-solution (availability....)
-Nicotine-solution for e-cigarrettes
-There was one more, I think it was PVAC = white wood glue, not sure, have to ask

Bob


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PostPosted: Sat May 25, 2019 9:54 pm 
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Hi Charlie g, Sauerkraut and Bob,

Osmium Tetroxide is a lethal poison, and in addition, can cause blindness.
here is a rational for charllie g's protocol: the viscosity of a 2% solution of methyl cellulose in water is ~400 cp. That of water is just ~1 cp. Dilution of the 2% MC solution, first by 2 (to make 50%) then by 67 (to make 1.5%) yields a viscosity of ~3. So it will slow down the cilliate motion by a factor of ~3 - very reasonable in my opinion. 2% MC is being sold as food additive and for other commercial purposes. It is very safe to use.

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PostPosted: Sun May 26, 2019 5:19 am 
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Hobbyst46 wrote:
Hi Charlie g, Sauerkraut and Bob,

Osmium Tetroxide is a lethal poison, and in addition, can cause blindness.
here is a rational for charllie g's protocol: the viscosity of a 2% solution of methyl cellulose in water is ~400 cp. That of water is just ~1 cp. Dilution of the 2% MC solution, first by 2 (to make 50%) then by 67 (to make 1.5%) yields a viscosity of ~3. So it will slow down the cilliate motion by a factor of ~3 - very reasonable in my opinion. 2% MC is being sold as food additive and for other commercial purposes. It is very safe to use.

Here is a link to show that it does exist and is not overly expensive.

https://www.homesciencetools.com/produc ... ose-30-ml/

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PostPosted: Sun May 26, 2019 5:58 am 
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As far as I know methyl cellulose is what is used to put wallpapers up the wall, you get it in DIY or decorating markets.

Bob


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PostPosted: Sun May 26, 2019 6:34 am 
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MicroBob wrote:
As far as I know methyl cellulose is what is used to put wallpapers up the wall, you get it in DIY or decorating markets.

Bob
So it is. It is a matter of quantity and convenience.

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PostPosted: Sun May 26, 2019 10:23 am 
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Note: buying a bottle of methyl cellulose solution is a lot more convenient than buying the powder and dissolving in water. It is soluble in cold water (NOT hot water), but only after prolonged stirring.

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PostPosted: Sun May 26, 2019 12:03 pm 
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Note that, as used in wallpaper paste, it typically has a fungicide added.
Tom


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PostPosted: Sun May 26, 2019 12:32 pm 
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tgss wrote:
Note that, as used in wallpaper paste, it typically has a fungicide added.
Tom
That will really slow them down!

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PostPosted: Tue Jun 04, 2019 12:34 pm 
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Hobbyst46 wrote:
Note: buying a bottle of methyl cellulose solution is a lot more convenient than buying the powder and dissolving in water. It is soluble in cold water (NOT hot water), but only after prolonged stirring.

I prepared 1.5% solution in water and and after a few days of stirring it seems to be all in solution but I see a lot of tiny dots/fibers floating around. Any idea how to get rid of them?


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PostPosted: Tue Jun 04, 2019 1:38 pm 
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Wes wrote:
Hobbyst46 wrote:
Note: buying a bottle of methyl cellulose solution is a lot more convenient than buying the powder and dissolving in water. It is soluble in cold water (NOT hot water), but only after prolonged stirring.

I prepared 1.5% solution in water and and after a few days of stirring it seems to be all in solution but I see a lot of tiny dots/fibers floating around. Any idea how to get rid of them?
I am afraid filtration is the only way. The ideal filter would be a disposable syringe filter, that looks like a top, and has Luer tips. It is a standard item in laboratories. One fills the syringe with the raw polymer solution, attaches the filter onto the syringe tip and pushes the syringe plunger slowly.
An the absence of such syringe filter, one may try gravity filtration through a funnel of coarse filter paper, or cheese cloth, coffee filter; a piece of ladies nylon stocking (high Denier) might help as well, perhaps.

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PostPosted: Tue Jun 04, 2019 2:15 pm 
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Hobbyst46 wrote:
Wes wrote:
Hobbyst46 wrote:
Note: buying a bottle of methyl cellulose solution is a lot more convenient than buying the powder and dissolving in water. It is soluble in cold water (NOT hot water), but only after prolonged stirring.

I prepared 1.5% solution in water and and after a few days of stirring it seems to be all in solution but I see a lot of tiny dots/fibers floating around. Any idea how to get rid of them?
I am afraid filtration is the only way. The ideal filter would be a disposable syringe filter, that looks like a top, and has Luer tips. It is a standard item in laboratories. One fills the syringe with the raw polymer solution, attaches the filter onto the syringe tip and pushes the syringe plunger slowly.
An the absence of such syringe filter, one may try gravity filtration through a funnel of coarse filter paper, or cheese cloth, coffee filter; a piece of ladies nylon stocking (high Denier) might help as well, perhaps.

I tried that. It was impossible, not even a single drop came out and I pushed to the limit of the syringe/filter. I ended up spinning it very fast which made a ton of difference, I could see all the crap pelleted at the bottom and a very nice clear solution on top which I collected and placed in a separate vial. It seems clean looking at it through the scope.

In terms of sample preparation is it a 50/50 mix of sample water with protozoans and methyl cellulose solution?


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PostPosted: Tue Jun 04, 2019 3:21 pm 
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Wes wrote:
Hobbyst46 wrote:
Wes wrote:
I prepared 1.5% solution in water and and after a few days of stirring it seems to be all in solution but I see a lot of tiny dots/fibers floating around. Any idea how to get rid of them?
...

I tried that. It was impossible, not even a single drop came out and I pushed to the limit of the syringe/filter. I ended up spinning it very fast which made a ton of difference, I could see all the crap pelleted at the bottom and a very nice clear solution on top which I collected and placed in a separate vial. It seems clean looking at it through the scope.

In terms of sample preparation is it a 50/50 mix of sample water with protozoans and methyl cellulose solution?
Congratulation on the successful centrifugation. Yes, this dilution is charlie g's protocol AFAIK.

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PostPosted: Tue Jun 04, 2019 4:36 pm 
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Wes wrote:
I ended up spinning it very fast which made a ton of difference, I could see all the crap pelleted at the bottom and a very nice clear solution on top which I collected and placed in a separate vial. It seems clean looking at it through the scope.


Hi Wes,
so you first centrifugated first and then filtered with the syringe filter Doron has suggested?

Hi Doron,
can you measure the mesh size of these filter tips for me and maybe post a link to them?
I have never had much contact to laboratory equipment and don't know what nice things are used there.


I always like recipes for microscopy material that is widely available. There remain so many special ingredients anyway and I have the impression that this difficult availability is often what keeps people from exploring new microscopy topics.


Bob


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PostPosted: Tue Jun 04, 2019 5:19 pm 
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MicroBob wrote:
Wes wrote:
I ended up spinning it very fast which made a ton of difference, I could see all the crap pelleted at the bottom and a very nice clear solution on top which I collected and placed in a separate vial. It seems clean looking at it through the scope.


Hi Wes,
so you first centrifugated first and then filtered with the syringe filter Doron has suggested?

I only centrifuged. I didn't see the need for filtering after the centrifugation step because there was nothing visible in the solution under high magnification i.e. clear solution was obtained. I doubt filtering would've worked after the centrifugation step but then again I used a 0,2 µm filter so it might work with a less stringent filter.


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PostPosted: Tue Jun 04, 2019 6:24 pm 
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MicroBob wrote:
Wes wrote:
I ended up spinning it very fast which made a ton of difference, I could see all the crap pelleted at the bottom and a very nice clear solution on top which I collected and placed in a separate vial. It seems clean looking at it through the scope.


Hi Wes,
so you first centrifugated first and then filtered with the syringe filter Doron has suggested?

Hi Doron,
can you measure the mesh size of these filter tips for me and maybe post a link to them?
I have never had much contact to laboratory equipment and don't know what nice things are used there.
I always like recipes for microscopy material that is widely available. There remain so many special ingredients anyway and I have the impression that this difficult availability is often what keeps people from exploring new microscopy topics.
Hi Bob,
Here is a photo of the filter. They are sold in quantities, I only have a few. It is a polymeric membrane - nylon, polycarbonate or other (possibly Teflon as well, although I have never seen one), diameter of about 25mm. within a plastic fixture, with Luer (syringe) connectors on both sides. There are other sizes and variants. The polymer is a very uniform mesh, and hole diameter is most often 0.2 or 0.45 micrometers (there are other hole sizes). The mesh size is hence printed on the filter and package, as hole diameter. One popular application is sterilization, since they can filter off microbes from a liquid. Sold by all chemical/biochemical/biological lab suppliers.
I had thought that the issue posted by Wes could be solved with such membrane filter, but apparently the viscosity of his cellulosic solution was too high. So, a very high pressure on the syringe plunger. According to my experience, very high pressure might break through the filter and destroy it, so the filtrate is the same as the unfiltered solution. But it depends on the liquid in question. With cellulosic materials, the viscosity rises steeply with concentration.
A centrifuge in such a case is a very good idea.

I too prefer home-made and easily available over lab-supply chemicals and accessories, but sometimes there is no choice...


Attachments:
Syringe filter.jpg
Syringe filter.jpg [ 99.75 KiB | Viewed 2227 times ]

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PostPosted: Tue Jun 04, 2019 6:53 pm 
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I've got a hand crank centrifuge for two quite big glasses, ca. 15x100mm. This will probably do. Otherwise we have an electric one in our group lab available.
Centrifuging is a nice process step, not messy, no fumes and no waste.

Thank you for showing these filters, Doron! I have heard of membrane filters but have not seen on in person. For an outdoor holiday in wilderness I once looked for water purification methods and there a pumps which probably employ such a filter, only bigger.
My idea was whether my 25µ stainless mesh might do but 0,xµ is a completely different thing. There are probably quite a few experiments that can be done with such a filter, e.g. removing pigments from fluids. Such a membrane could also separate inhabitants in a plancton aquarium while allowing exchange of oxygen and minerals.
With a thin syringe one can produce quite a pressure, I can imagine that even the filter housing could be crackes apart if one would really try.
As primary school boys we did do target shooting with syringe needles, powered by the compressed air from thin syringes... No recommended safety toys! :shock:

Bob


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PostPosted: Thu Jun 06, 2019 11:04 pm 
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Location: Oregon, USA
Here’s a YouTube link that compares methylcellulose to physillium seed husk.

https://m.youtube.com/watch?v=vowfgDu8Fz4

Think I’ll give the seed husks a try and see how that goes.

Heather


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