Better choice of mounting medium for phase contrast

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daruosha
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Better choice of mounting medium for phase contrast

#1 Post by daruosha » Sat Feb 15, 2020 9:52 pm

The title seems self-explanatory, however let me explain myself:

I wanted to observe non-stained cheek cells with phase contrast and tried differenet mounting mediums:
1- Glycerol: I didn't like the results, the contrast was not that much and it was very difficult to see cells nuclei. Since Glycerol has a higher refractive index than water (1.4731 vs 1.33), I accepted the result and moved on.
2- Water: After glycerol failure, I tried water as mounting medium and as expected the results were much much better, however when I dropped immersion oil on the coverslip, focusing with 100x objective was a nightmare, as soon as the oil touches the objective, surface tension between oil and coverslip messes with focus and you have to focus and refocus several times to keep the coverslip in place and achieve a consistent focus. I never had this problem with other mountants like glycerol or entellan or NPM.
3- Entellan: Considering even higher refractive index, I didn't expect any good results and I was right. I heat fixed the cells on the slide and let them dry, afterward a drop of Entellan and a coverslip on top. The results were mediocre (as i expected).

I understand how the phase contrast works and how it relies on differences between refractive indices and so on, but I'm wondering what is the best permanent mounting medium for phase contrast observation? I'm planing to make some diatom slides and people suggest mediums with even higher refractive index like Pleurax or Hyrax. What do you suggest? Somebody please enlighten me.

I can take new pictures with each mounting medium if it helps.

Thanks.
Daruosh.

MicroBob
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Re: Better choice of mounting medium for phase contrast

#2 Post by MicroBob » Sun Feb 16, 2020 7:25 am

There are several requirements that a mountant has to fulfill:
- it has to behave well with the specimens, especially when these are fresh,
- it has to give the right amount of contrast between mountant and specimen
- it has to behave well with the used stains
- it shouldn't damage the specimen due to shrinking
- it has to hold the cover slip well
- it has to give the needed durability

Since you look for contrast between the nucleus and the surrounding cell material the mountant's r.i. is probably not that important.

Here is a link to a recipe: http://up.picr.de/12334113bo.pdf

Hobbyst46
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Re: Better choice of mounting medium for phase contrast

#3 Post by Hobbyst46 » Sun Feb 16, 2020 9:22 am

daruosha wrote:
Sat Feb 15, 2020 9:52 pm
I understand how the phase contrast works and how it relies on differences between refractive indices and so on, but I'm wondering what is the best permanent mounting medium for phase contrast observation? I'm planing to make some diatom slides and people suggest mediums with even higher refractive index like Pleurax or Hyrax. What do you suggest? Somebody please enlighten me...
There are several implicit notions within this line of thought.
1. Phase contrast is a method to increase the contrast between the various regions of the specimen and between the specimen and its surroundings, whatever the RI of the mountant. The result does depend on the RI of the mountant but the important contribution to contrast arises from the phase differences due to the illuminating beam.
Thus, phase contrast is effective even when the cheek cells are mounted in water, so the RI difference is very small.
Likewise, phase contrast is effective for diatoms in water, again in spite of the small RI difference.
Furthermore, one advantage of phase contrast is that it is non-destructive, so effective for live, non-fixed specimens. Cells, diatoms and protists.
2. To preserve a live specimen for a permanent slide, fixation is needed. Mounting in water will not preserve a live specimen for long.
3. As mentioned above, the effect of phase contrast can depend on the mountant, to some extent.
4. Using high RI mountants for diatoms preceded phase contrast by many decades. High RI mountants were adopted to increase the contrast between the diatom and the surroundings in brightfield, before phase contrast was discovered. When a diatom is mounted in a high RI resin such as Naphrax or Pleurax or Styrax or Aroclor... it is well contrasted and resolved under ordinary bright field illumination.
5. Most non-water mountants will change the form of a live specimen, not just "stabilize" it but cause shrinkage, deformation etc. The preparation of specimens is often specific and adapted for that particular specimen.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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daruosha
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Re: Better choice of mounting medium for phase contrast

#4 Post by daruosha » Sun Feb 16, 2020 10:09 am

The rate if information delivery is amazing. I appreciate your answers. Is it safe to conclude there's not an specific/preferred mountant for phase contrast?

The higher RI, the better contrast for bright field, yes but I'm looking for a permanent mountant with RI close to the water.
Daruosh.

Hobbyst46
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Re: Better choice of mounting medium for phase contrast

#5 Post by Hobbyst46 » Sun Feb 16, 2020 10:51 am

daruosha wrote:
Sun Feb 16, 2020 10:09 am
..Is it safe to conclude there's not an specific/preferred mountant for phase contrast ? The higher RI, the better contrast for bright field, yes but I'm looking for a permanent mountant with RI close to the water.
I would choose the mountant according to the guidelines given by MicroBob above.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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daruosha
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Re: Better choice of mounting medium for phase contrast

#6 Post by daruosha » Tue Feb 18, 2020 2:41 am

Hobbyst46 and MicroBob,

Thanks for your comments and tips. Your contribution to the forum is truly amazing. I am an EEVBlog forum addict, but here in this forum I feel at home. :)
Daruosh.

apochronaut
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Re: Better choice of mounting medium for phase contrast

#7 Post by apochronaut » Tue Feb 18, 2020 11:13 am

Essentially, you are trying to come up with a thicker water. This has been an area of interest for many years in microscopy but also in other disciplines where water is of prime importance in a finished product, yet it's "wateriness" is too much. In the food industry, mouth feel is dependent on the water content of many foods becoming thicker and therefore coating sensory receptacles of the mouth more evenly. That's why there are so many emulsifiers and thickeners in processed foods. Food grade gels and thickeners might likely hold the silver bullet of your quest. Possibly emulsifiers but they usually function to meld oil and water.

I'm not sure whether this is of much value to you but I will pass it along. You may already know as much as is relevant here.

http://www.ihcworld.com/_protocols/hist ... medium.htm

but then there is also this. you might do a search of some other gums and thickeners too. I use pectin some.

https://www.researchgate.net/figure/Ref ... _282861340

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daruosha
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Re: Better choice of mounting medium for phase contrast

#8 Post by daruosha » Tue Feb 18, 2020 2:30 pm

The second link is very helpful. I'm doing some experiments and will come up with my results. It seems I'm getting ahead of myself.
Daruosh.

mnmyco
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Re: Better choice of mounting medium for phase contrast

#9 Post by mnmyco » Wed Feb 19, 2020 2:17 am

Try a saturated solution of calcium chloride and/or saturated solution of potassium acetate. The CaCl2 won’t dry out. Glycerol saturated with sodium borate (20 mule team borax) is nice but annoying to make. Pure wintergreen oil. Water saturated with thymol, which is insanely fragrant and not pleasant. These are all for temp slides. If I recall any others I will add them.

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