Hello All,
I had been having problems with Euparal mounts going cloudy. I think I have worked out the problem and certainly I no longer have the problem.
The problem: (Sorry not best image)
Now look at my set up:
I had been using those crazy teapots as wash "capsules" to use Walter's parlance.
I had used them with whole mount fructose and had some success.
They are handy as the handle bit is good for pipette resting/forceps resting etc.
I was using warm water fixed onion.
I was also using ethanol not propan-2-ol (I'm a science teacher - I use IAPAC, I mean one of my students might read this
I was also using ethanol to clean my slides.
What I think was going wrong:
a) The onion wasn't being deydrated properly as it wasn't fully submerged during 25%,50%,75%,100% drying.
b) My slides still had ethanol on them (slides wet with etoh) when I put the not properly dried onion skin on them.
c) The ethanol evaporates but tends to leave some water hanging around
--> hence the euparal goes cloudy. Sure Euparal can handle a little water but not much.
Now I didn't just change on thing but hey I wanted nice slides.
Changes:
1) Onion skin now in FAA (my own homebrew made from 40% formalin from aquarium shop 15ml, distilled domestic vinegar 40ml, ethanol (96%) 45 ml)
2) Propan2-ol used for dehydration. I figured I would do as John B did and that perhaps ethanol with its slightly lower boiling point might lead to more water issues. I also used more normal vessels (as in 30 ml wide mouthed glass jars) and made sure the onion was submerged
3) I took more care with drying my slides. I still use ethanol for this but I went to town making sure they were good and dry.
Happily it worked.
Here are some pics
Would people agree that these are plasmadesmata?
Any comments welcome
Problems with Euparal mounts going cloudy and solutions
Re: Problems with Euparal mounts going cloudy and solutions
Super job Bill, lovely clean mounts. I'd certainly agree re the plasmodesmata - bang-on I'd say.
Fascinating to ponder that the plasmodesmata aren't 'holes' in the plasma-membrane, but may rather be considered 'unpinched' channels where the cell-walls do not actually isolate cells but 'squeeze them into packets' that are then able to exhibit a degree of specialization or even autonomy, but are actually elements of the vast continuum of the plant body, all connected - the plasma-membrane unbroken by these selectively-permeable conduits/channels (desmotubules?)! This fascinates me as it affords several ways of conceptualizing a plant's structure - the "a plant makes cells the cells don't make the plant" position?
Pleased you sorted the Euparal out OK - it's really a case of keeping it simple and consistent with your setup I think - you've yet to get your work-flows established that's all. I went through this stage (and still do with each new technique I try to use) and it soon becomes clear where the inefficiencies and bottlenecks are - many, many times it's a simple case of positioning things where you can reach them once the process begins!
I'd stick to IPA for everything OH if I were you, it's tried, tested and consistent - the azeotrope it forms is easily within the limits of Euparal's tolerance in my experience. Remember to seal your fine mounts after about a week, ordinary nail-polish does a good job - I have all my 'Walter-D' slides safely intact by these methods.
Really nice series Bill, keep up the good work!
p.s. I soon discovered that I needed tissue pieces far smaller than I was using at first, which also makes them easier to process and mount - about 5mm as a largest dimension is a good size you may find. Excellent mounts and crisp colourful images!
Fascinating to ponder that the plasmodesmata aren't 'holes' in the plasma-membrane, but may rather be considered 'unpinched' channels where the cell-walls do not actually isolate cells but 'squeeze them into packets' that are then able to exhibit a degree of specialization or even autonomy, but are actually elements of the vast continuum of the plant body, all connected - the plasma-membrane unbroken by these selectively-permeable conduits/channels (desmotubules?)! This fascinates me as it affords several ways of conceptualizing a plant's structure - the "a plant makes cells the cells don't make the plant" position?
Pleased you sorted the Euparal out OK - it's really a case of keeping it simple and consistent with your setup I think - you've yet to get your work-flows established that's all. I went through this stage (and still do with each new technique I try to use) and it soon becomes clear where the inefficiencies and bottlenecks are - many, many times it's a simple case of positioning things where you can reach them once the process begins!
I'd stick to IPA for everything OH if I were you, it's tried, tested and consistent - the azeotrope it forms is easily within the limits of Euparal's tolerance in my experience. Remember to seal your fine mounts after about a week, ordinary nail-polish does a good job - I have all my 'Walter-D' slides safely intact by these methods.
Really nice series Bill, keep up the good work!
p.s. I soon discovered that I needed tissue pieces far smaller than I was using at first, which also makes them easier to process and mount - about 5mm as a largest dimension is a good size you may find. Excellent mounts and crisp colourful images!
John B
Re: Problems with Euparal mounts going cloudy and solutions
Ah, Bill, almost forgot - onion-peels are troublesome in water as you know, they don't like it!
This is my quick & cheap 'vacuum-machine' and it works perfectly for any type of immersion/infiltration - remember to release the vacuum a couple of times then re-apply, helps fluid to flow into the tissue which can collapse as air leaves if you forget this step...
Works very well indeed - very satisfying to watch your tissue sink as the bubbles leave! Good luck, keep us informed of your adventures!
This is my quick & cheap 'vacuum-machine' and it works perfectly for any type of immersion/infiltration - remember to release the vacuum a couple of times then re-apply, helps fluid to flow into the tissue which can collapse as air leaves if you forget this step...
Works very well indeed - very satisfying to watch your tissue sink as the bubbles leave! Good luck, keep us informed of your adventures!
John B