toluidine blue

Ross Koning

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Oct 8, 1997, 3:00:00 AM10/8/97

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At 8:35 AM -0400 10/8/97, Wilson Taylor wrote:
>Hello all,
>
>I use a simple aqueous solution of Toluidine Blue (0.2%) and have very good
>results with lignified cell walls (blue or blue green) (thanks to Sue Schen=
k
>for the lead and recipe - it beats phloroglucinol/HCl by a long shot!)
>
>Does anyone know of any advantage to the recipe below? What is the aqueous
>sodium tetraborate for?
>
>Other suggestions for simple aqueous stains for fresh material would be
>welcome (Let's get some ANATOMISTS talking to one another here instead of
>all of those darned physiologists [tongue firmly in cheek])

Hi Wil!

I second your suggestion (and I'm a physiologist!).

I have used the simple aqueous Tol blue solution and my students
get fairly good results...but NEVER beating phloroglucinol/HCl.

The secret to Phloroglucinol is making the phloroglucinol
in an alcoholic solution. I use HCl in a separate dropper
bottle diluted 1 conc HCl to 4 parts water (resulting in about
10% HCl final conc). Apply the two drops to a specimen and
in about 10 seconds the results are spectacular. For long-term
storage and classroom use, I prefer Nalgene dropper bottles
with unitary caps. You never get more than one drop at a time
and nothing degrades in the acid.

What I don't like about Phloroglucinol is the corrosion you can
get on mechanical stages and the tips of objective lenses if
the students aren't meticulous with the acid...and promptly
removing the slides from the stages before storing the microscopes.
I'm excited to try the tetraborate addition to see if it improves
the contrast with Tol blue...that would be nice and might convince
me to switch away from Phloroglucinol. The tol blue w/o borate
is just too murky to me.

ross

_______________________________________________________________
Ross Koning | kon...@ecsu.ctstateu.edu
Biology Department | http://koning.ecsu.ctstateu.edu/
Eastern CT State University | phone: 860-465-5327
Willimantic, CT 06226 USA | fax: 860-465-4479
____________________________|__________________________________

Electronic services composed and served from =95Macintosh hardware.

Douglas P. Jensen

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Oct 8, 1997, 3:00:00 AM10/8/97

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At 07:03 AM 10/8/97 -0700, Ross Koning wrote:
>[lots snipped out]

>What I don't like about Phloroglucinol is the corrosion you can
>get on mechanical stages and the tips of objective lenses if
>the students aren't meticulous with the acid...and promptly
>removing the slides from the stages before storing the microscopes.
>I'm excited to try the tetraborate addition to see if it improves
>the contrast with Tol blue...that would be nice and might convince
>me to switch away from Phloroglucinol. The tol blue w/o borate
>is just too murky to me.
>
>ross
>

I partially get around the corrosive properties of HCl by having the
students destain in water. we stain in syracuse dishes. After staining, we
put sections in another dish with water. The results are great and I don't
have to worry about people getting acid burns, stains, or leaving slides
under the scopes.
The results are fine.

Doug

Douglas P. Jensen, Assistant Professor of Biology
PO Box 9615
Hollins College
Roanoke, Virginia 24020
(540)362-6549
dje...@hollins.edu

Wilson Taylor

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Oct 8, 1997, 3:00:00 AM10/8/97

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Hello all,

I use a simple aqueous solution of Toluidine Blue (0.2%) and have very good

results with lignified cell walls (blue or blue green) (thanks to Sue Schenk

for the lead and recipe - it beats phloroglucinol/HCl by a long shot!)

Does anyone know of any advantage to the recipe below? What is the aqueous
sodium tetraborate for?

Other suggestions for simple aqueous stains for fresh material would be
welcome (Let's get some ANATOMISTS talking to one another here instead of
all of those darned physiologists [tongue firmly in cheek])

Wil Taylor

>A general recipe for Toluidine Blue stain is:
> 0.01% Toluidine Blue (powder) in 10 mg
> 0.1% aqueous sodium tetraborate (sodium borate) 100 mg
> 100 mL water
********************************************************************************

Wilson A. Taylor
Department of Biology
University of Wisconsin-Eau Claire
Eau Claire, WI 54701
tayl...@uwec.edu

********************************************************************************

Frederique Guinel f

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Oct 9, 1997, 3:00:00 AM10/9/97

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Toluidine blue O is a polychromatic dye, i.e. it absorbs
different colours depending on the nature of its chemical binding with
the components of the tissue. It is positively charged at a pH of 4.4.
At this pH, it will bind to carboxylated polysaccharides such as pectins
in the cell walls and it will colour them pink. Compounds containing
benzene rings, such as lignin, will be coloured green (lignin in the
xylem tissue for example) or turquoise (lignin of the sclerenchyma). The
colour will depend upon the particular chemical structure of the compound.

At a lower acidic pH, toluidine blue gives only a blue or green
colour. The pectins at that pH do not carry negative charges since
their carboxyl groups would be complete (COOH); therefore they cannot react
with the dye. Only the lignin with its benzene rings will do so, hence
the green colour.

With toluidine blue at a higher basic pH (11.1), on the contrary, the
stained tissue is mostly dark pink since most (if not all) pectins will
be charged. This will hinder the green colour of the lignin. At this
pH, the dye penetrates easily the resin used to embed plant tissue. It
is quite useful to stain fixed tissue.

I hope this makes sense to you. You can find more about
Toluidine Blue in the book entitled "The study of Plant structure:
Principles and selected methods" by O'Brien and McCully, Termarcarphi Pty
Ltd, 1981.

One last thing: two weeks ago, I went to a marsh with my
students. We brought back leaves of cattails. These are easy to section
by hand since they are quite stiff. One can section transversally or cut
the leaves in half longitudinally. Stained with Toluidine blue (pH 4.4),
the sections were absolutely gorgeous. One could see parenchyma cells
anastomosing forming very large aerenchyma, and beautiful lignified tissues
(especially sclerenchyma). If you have the chance, loot at that same
tissue under a fluorescent scope, it is magnificent. I should stop now,
I could continue to talk about these leaves for quite a long time.

Have all a nice Fall term.

Frederique C. Guinel
Assistant Professor
Department of Biology
Wilfrid Laurier University
Waterloo
Ontario, Canada, N2L 3C5