Clearing Agents

[einman]

Initially while gathering materials to begin making my own slide mounts I had planned on using Xylene I have stored in the garage. It is readily available at any Lowes or Home depot. However the safety profile is less than desirable having a flash point of about 80F and is a health hazard. Other alternatives available are:

Histo-Clear
Histo-clear II
Americlear
Hemo-De
Masterclear
Clearity
to mention a few. The prevalence of alternatives to xylene can be explained by the fact they consist primarily of citrus D-limonene with some additional components.

While Histo-clear consists of 95-100% d-limonene and its low odor version, Histo-Clear II consists of 10-30% d-limonene mixed with about 70% unidentified aliphatic hydrocarbons.

This pretty much describes the other brands as well.

Has anyone tried Histo-Clear vs Histo-clear II?

Given the primary ingredient is D-limonene I thought might look at purchasing it on sites like Amazon where it is often promoted as a "cleaning" agent. Although there are many brands, the price is not really that much less than Histo-Clear or Histo-clear II.. You can still expect to pay about $60/gallon, although shipping tends to be less via Amazon than through a scientific supply house.

Any suggestions?

How many times can you re-use a typical 4 oz container of this material for prepping tissue?

[mrsonchus]

Hi einman, over here in the UK I pay £85 + VAT and carriage per US-gallon - a total of about £125 per US-gallon all-in!

One US-gallon (3.7L I think) lasts me between 1 year and about 8 months depending of course upon my activities. I've quite a few posts detailing the entire process that you may find helpful - just search by 'mrsonchus' and a few key words - I've lots on this forum that will help you begin.
Fell free to ask me directly here or in pm also - no trouble at all to take you through it to get you started.

Oh, I find the Coplin jars often allow slides to touch - awkward in use and very expensive to buy.
I use the superbly cheap trough+racks from China - they take up to 24 slides at a time - and you'll need at least a dozen, double that if you progress...
cheap and very good staining trough with rack

They are unaffected by alcohol, acetic acid, anything in fact including Histoclear.
John B

p.s. This is a book I bought back in Jan 2015 - the best current book there is I'd say - worth every penny.
Ruzin's microtechnique book

[einman]

Thanks so much.

The Coplin jars were quite cheap actually. Although I will check out the trays you suggest.

I have the Ruzin book. Bought it some time ago, after first reading your escapades, along with the following:

Methods in Plant Histology by Charles Chamberlain University of Chicago 4th edition 1930
Elements of Botanical Microtechnique John E Sass Iowa State College 1940
Plant Microtechnique Donald Alexander Johansen 1940

These books are quite old but I enjoy owning, browsing and learning from such old texts. The discussions on technique, reagents etc are incredible. Such a fascinating hobby. I could see how it could consume your time completely. One reason for hesitating. I tend to either go full speed or don't go at all. LOL

[mrsonchus]

Ha, I have them too - all excellent books and as you clearly realise, a good 90% of the techniques are still good for today's little-changed 'paraffin technique' in the essentials.
I often think of such sources as almost 'collateral sources' - so many quite chance lines written, or written in a certain way that suddenly illuminates almost an entire principle - for example the 'acid stain' 'basic stain' categories and their complementary but not entirely separate use..

I've always approached study from an understanding of first-principles rather than the 'try to remember it all' perspective.

Take a step back from Ruzin's really very long (in terms of stage duration) protocols to realise that most herbaceous tissue needs far less time at each stage than the 4hrs+ often written by Ruzin.

Start with a soft stem (stems are so much easier to orient when embedding in wax) tissue, say Daffodil flower-stem.

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 that helps a little -
Ruzin can make the process seem quite gargantuan - it's not easy, but it's not as difficult as Ruzin's book may suggest.

[billbillt]

Hi John,

Thanks for sharing a condensed list of procedures that you use... As always you post the most interesting items!..

BillT

[Culicoides]

Hi, JohnB,
Re your mounting protocol, do you know if histoclear is compatible with Canada balsam? I am looking for something that balsam ( that is natural balsam, not balsam thinned with xylene) can be mixed with, for mounting small insects that is:
Insect in isopropyl alcohol to histoclear to mount in balsam thinned with histoclear.
With your experience of histoclear, do you think this might work?
I hope you don't get too much snow in Cumbria over Christmas; amazingly we have missed the snow entirely down here in Guildford so far.
All good wishes, JOHN B.

[mrsonchus]

Hi John,

Thanks for sharing a condensed list of procedures that you use... As always you post the most interesting items!..

BillT

Hi Bill, thanks my friend, always good to hear from you.
John B.

[mrsonchus]

Hi, JohnB,
Re your mounting protocol, do you know if histoclear is compatible with Canada balsam? I am looking for something that balsam ( that is natural balsam, not balsam thinned with xylene) can be mixed with, for mounting small insects that is:
Insect in isopropyl alcohol to histoclear to mount in balsam thinned with histoclear.
With your experience of histoclear, do you think this might work?
I hope you don't get too much snow in Cumbria over Christmas; amazingly we have missed the snow entirely down here in Guildford so far.
All good wishes, JOHN B.

It's coming for you......

Hmm, I've no experience with natural balsam I'm afraid - but I'd be surprised if they were incompatible. Why not just use either 'Onimount' or 'Numount' (Numount is sold by Brunel in very reasonably-priced pots)?
Regarding Omnimount - it's not my favourite as I find it invariably seeps out of the edges of the cover-slip and gives a rather messy finish. It also takes absolutely ages to harden - over a month or even longer. This is when used with the superb Histoclear (original). Numount on the other hand is I find very good indeed - dries and hardens quickly (I'd say dry in about 1hr, hardened in about 2 days).

By 'dried' I mean stable enough to take an early look at the slide without fear of it losing it's cover-slip. By 'hardened' I mean stable enough to enable thorough cleansing of the slides without fear of damage - I usually use soapy water, cabinet air-dry and soft polish if necessary. It can only really be said that the slide is also at it's optical best at the hardening stage also.

Hope that helps a little.

John B.

[Culicoides]

Many thanks. That sounds worth giving it a try. I'll contact Brunel after the holiday. JOHN B

[einman]

Here is a website that offers multiple mounting options depending on your application.: https://www.emsdiasum.com/microscopy/pr ... media.aspx

It is just amazing the variety based on specific application.