Perennial milk-weed - rust-fungus slides

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coominya
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Re: Perennial milk-weed - rust-fungus slides

#61 Post by coominya » Wed May 09, 2018 2:17 pm

mrsonchus wrote:Dead-right my friend. We all have to start at the beginning. You'll find your technique will improve very quickly indeed - remember to keep the sample and the blade wet and 'slice' rather than chop if you can.
You'll be amazed what can be achieved with basic hand-sectioning!

Good luck, let us know how you get on.
Well I was happy with my first attempts Mr.S, I took a weed out of the garden and sliced it's stalk as you recommended and got some fairly thin sections which I explored. The detail in them was all the same, as to be accepted, and i had no idea what i was actually observing but it was very rewarding non the less. there is certainly a lot going on in there and some very complex structures. Thanks for your help, here are a couple of pix, sorry for the thread hijack ;)

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billbillt
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Re: Perennial milk-weed - rust-fungus slides

#62 Post by billbillt » Wed May 09, 2018 5:41 pm

Great work.. I used to know the name of the "spring" shaped objects, but I have forgotten.. They are very commonly photographed..

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MikeA
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Re: Perennial milk-weed - rust-fungus slides

#63 Post by MikeA » Sat May 19, 2018 10:27 am

Hello All,

Spring has finally (!) arrived in NE Ohio and the young plants are popping up everywhere. I have harvested a number of samples that are held 50 ml bottles of FAA in the refrigerator at ~4°C, so ready to progress to next steps.

In searching for alternatives to the hard to obtain/pricey materials, I came up with some interesting information - one write up (cannot remember from where) clearly states IPA or Denatured Methanol may be used wherever Ethanol is noted, which basically endorses what has been said here re using IPA. I bought some 99% IPA, as well as a supply of denatured methanol so will be using one of those in lieu of the called for EtOH.

Another alternative to xylene as a clearing agent is "CitriSolv™ Hybrid Solvent and Clearing Agent, but it is
only available on GSA/VA Contract for Federal Government customers only."

A review of the SDS indicates a high proportion of d-Limonene in this product, and as it turns out, Histoclear SDS lists its components as 95 - 100% d-Limonene; Histoclear II is rather different, with 70 - 90% Heavy Naphtha (CAS 64742-48-9) and 10 - 30% d-Limonene. d-Limonene has been shown to be a safe and effective alternative to Xylene and is fully biodegradable - it is derived from the peels of citrus fruit. (I expect prices from various suppliers may reflect seasonality of fruit.) I happen to have a gallon of d-Limonene sitting in my supply, so will be using it for clearing prior to infiltration with Paraplast Plus.

I do have some questions: does anyone have a protocol for replacing the various materials/dilutions, e.g. how long can one use the FAA, or more correctly, how many samples can be processed through it? Are the higher concentrations of water/OH moved down after x-many samples? How is the water content of the ROH measured as samples are passed through? I am guessing they get used for quite some time with limited samples going through them, but curious nevertheless.
All the best,
Mike
'Nil Satis Nisi Optimum'

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mrsonchus
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Re: Perennial milk-weed - rust-fungus slides

#64 Post by mrsonchus » Sat May 19, 2018 12:54 pm

Aha coominya - how I haven't commented 'til now on your splendid pics I don't know!

You've caught an oblique angle across the stem which has, as it also does with processed microtome sectioning, revealed a 3D aspect to us of the Xylem vessels!
The tubular nature of the Xylem - the water-highways of a plant, is clear to see here. Think of the spiral of wire embedded, in clear plastic of your vacuum-cleaner's extension pipe - very nearly the same structure, excepting that in the plant's Xylem the reinforcing spiral is on the surface of (but not actually originating from) what would be the plastic - the primary cell-wall in this case.

As you say, so much 'goes on' when we look closely at plants; this together with their easily-studied form/function aspect, let alone the taxonomic/systematic structures/characters provides us with a truly stunning and mind-bogglingly fascinating source for study, adventure and wonder!

Congratulations on a great start to your explorations - a good lesson also in your images, which is that perfectly transverse (in this context meaning perpendicular to the organ's surface) sections are by no means exclusively the sections that are of great use when exploring!

Well done, let's have a few more my friend - indulge yourself and, as I am doing today in our sunny gardens, 'go quite wild'!

John B. :)
John B

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mrsonchus
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Re: Perennial milk-weed - rust-fungus slides

#65 Post by mrsonchus » Sat May 19, 2018 1:16 pm

MikeA wrote:Hello All,

..............
I do have some questions: does anyone have a protocol for replacing the various materials/dilutions, e.g. how long can one use the FAA, or more correctly, how many samples can be processed through it? Are the higher concentrations of water/OH moved down after x-many samples? How is the water content of the ROH measured as samples are passed through? I am guessing they get used for quite some time with limited samples going through them, but curious nevertheless.
Hi Mike, well, that's a tricky question, one that is truly variable depending upon many factors.... But, some idea must be adopted of course!
Personally I always use fresh (unused and not more than about 1 month since mixing) FAA in sample jars with each new batch of tissue - in which they may stay practically indefinitely preserved (and indeed in a suitable state to process further whenever you wish).

As for the dehydration series, I also mix and use these fresh with each batch of tissue - the use of 'just the right size' of jar for your samples/cassettes etc mitigates greatly any waste I find. The cheapness of the IPA and DIW components makes me quite happy to adopt this approach.

As for OH concentrations - they absolutely don't need to be measured beyond the w/w accuracy with which they are initially mixed. The only thing to be sure of is that the 'pure' stage is indeed of unused (probably nominally 95% which is perfectly sufficient to use as 'pure' IPA) OH straight from the bottle as it were.

The Histoclear or whichever clearing-agent you use, probably, will be OK to use several times (makes sense also considering it's hideously high cost!) - it's job is to remove the (pure) OH from the final dehydration stage by displacement and to go-on to be the ante-medium for wax infiltration that follows. I've also trialled processing without any 'clearing' stage - i.e. no use of Histoclear, simply letting the OH (IPA) funtion as the wax ante-medium with complete success.
Histoclear will still be needed as a de-waxing agent when the sections, dried onto slides, are to have the wax that infiltrates them removed prior to staining and mounting of course...

My advice - keep processing-container (jars...) volumes no larger that necessary and use fresh chemicals every time - the cost is pretty low.

Hope this helps a little my friend.

John B. :)
John B

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mrsonchus
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Re: Perennial milk-weed - rust-fungus slides

#66 Post by mrsonchus » Sat May 19, 2018 1:16 pm

URK - sorry - seem to have posted twice! :oops:

John B.
John B

Fer961118
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Re: Perennial milk-weed - rust-fungus slides

#67 Post by Fer961118 » Sun Jan 03, 2021 4:56 pm

Hi John,
I loved your histological slide preparations; I’ve been working on some fugal structures, as rust teliospores, pseudothecia from Ascomycota among others, can you share your recipe for the staining, I’m very impressed with your results about the use of the Orange G.

Best regards,

Fernando

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