Embeding problem, glycerine jelly update and stain sensitivety.

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GrbovicMicroscopy
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Embeding problem, glycerine jelly update and stain sensitivety.

#1 Post by GrbovicMicroscopy » Sun Sep 01, 2019 3:12 pm

Hello
So i tried to embed the stem of Petroselinum crispum in paraffin wax, it did not work as the wax did not penetrate the cells so the stem shrunk after a while. I did fixate it in achohol then embeded it. What do i need to soak (fix) the specimen in to get a nice embedment i would like to keep the specimen around?

As for the glycerin jelly. It is very nice as i avoid using Canada balsam and Xylol so less danger. I do need to get a heat pad or something like that as my little oil candle i made ,using a 5ml glass bottle which i use for specimen storing in alchohol, some aluminium foil and a wick, overheats the jelly and bubbles start forming.


An observation i made while heating the jelly and placing the specimen inside was that the stain Safranin seems to bee heat sensitive and desolves into the jelly.. On the other hand Astra blue seem to be more stabile at those temperatures.

MicroBob
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Joined: Sun Dec 25, 2016 9:11 am

Re: Embeding problem, glycerine jelly update and stain sensitivety.

#2 Post by MicroBob » Sun Sep 01, 2019 3:56 pm

Hi,
for the embedding protocol you might google for Iso-Methode Klaus Henkel. This is comparatively amateur friendly.
On our website you can find proven recipes too: http://www.mikrohamburg.de/Tips/Paraffinschnitte_02.pdf

For careful heating I use:
- a baby bottle warmer :D
- an aluminium block with heating resistor and temperature feeler and a PID-regulator and laptop power supply. Today I would use the heat element for 3d-printers, 12V 40W.

Stains and pH value of the mounting media have to match, otherwise the stain is drawn out over time.

Bob

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mrsonchus
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Location: Cumbria, UK

Re: Embeding problem, glycerine jelly update and stain sensitivety.

#3 Post by mrsonchus » Sun Sep 01, 2019 7:47 pm

Hi, for a gentle heat such as that needed for glycerin jelly, I use a little coffe-cup warmer, cheap and very convenient.

As far as the wax embedding goes, I'm afraid to say that the entire process is a long one. The production of a permanently stained & mounted slide involves many multi-stage procedures, the first of which is fixation, then dehydration, infiltration (where molten-wax is infiltrated into the tissue right down to the intra-cellular level) then embedding. Then there's the sectioning, de-waxing, staining and mounting to be completed....

I started from the very beginning and if you search this forum for my posts, going back to 2015 when I first started, you'll find my posts detailing every single stage of the entire process, from plant in the ground to several-micron thick stained and permanently mounted sections on microscope slides.

Here is the process, copied from one of my earlier posts see this post which includes the whole process listed
Stages in a nutshell,


kill it (FAA-50 for >=24hrs)
dehydrate it (Isopropanol series)
remove alcohol with wax-antemedium or 'clearing agent' - (Histoclear)
infiltrate with molten wax
embed in wax
section from wax-block
de-wax with wax solvent (Histoclear)
remove Histoclear with alcohol
remove alcohol with water
stain with aqueous stain such as Safranin
dehydrate with alcohol
stain with alcohol-base stain such as Fast-green
remove alcohol with Histoclear (Histoclear is compatible with resinous mountant such as 'Numount' of 'Omnimount')
mount in resinous mountant

Hope this helps a bit.
John B

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