Re: Toludine Blue O - Metachromasia - pH - AND Science

From:"Monson, Frederick C."

Morning All,
	
	For those among you who are wise, you should probably immediately
jump or GOTO END!

	This is difficult to put into words without chancing a gross error
or causing some offence, but here goes anyway.  

	When we "fix" a specimen taken from a biological source, we NEVER
quite know what we are doing.  The entire process is 'outcome-based'
(Ugh!!!).  How does it look?  Does the fixative 'coagulate' or 'cross-link'
the myriad of chemical entities within the sample?  What is the effect of
specimen size?  What about temperature?  How long to kill?  How long to
achieve minimum preservation?  Preservation of what?  What will constitute
statistically comparable samples that permit comparison of specimens of
mouse and elephant liver?  We preserve or fix or kill or maintain color or
get rid of it.  Behind every act there lies a reason, and in consequence of
every act there is produced an uncountable number of variables which only
REPRESENT what existED in the original, living, organism.  Everyone says
that s/he already knows this!

	Metachromasia is part physical chemistry, part biochemistry (and by
reason of those two, for many, little more than pure magic!).  Metachromasia
has been extensively used, extensively studied and extensively reported
upon.  Several items of information are clear [This may be an
exaggeration!].

	1.  The polychrome effect noted in the use of several related dyes
of the thiazine (quinone-imine/methylene blue) family is based on the
polymerization of dye molecules and the distances between adjacent acidic
groups on the substrate.

	2.  A loss of the polychrome effect could be attributed to*:
		a.  decrease in the concentration of some tissue/substrate
component
		b.  decarboxylation [- or - esterification with alcohol?]
		c.  degradation of the substrate to diffusible constituents.

	    [*   see Pearse, A.G.E.(1985), Histochemistry, Theoretical and
Applied(4th Ed.), Vol II, Churchill Livingstone, NY,NY, pp701-710.  ISBN:
0-443-02997-0.

	3.  In order for the metachromatic color. for (Toluidine Blue O,
that is purple [red + blue]) to be observed, the appropriately spaced acid
moieties must be deprotonated, i.e. negative in charge.  Otherwise, the
orthochromatic color is observed.  

	In other words, in order for there to be metachromasia of a
particular component of the tissue, that component must have appropriately
spaced negatively charged groups.

	4.  At pH 7, most 'acid' moieties in biological systems are
deprotonated, and thus, negatively charged.  The isoelectric points of most
macromolecules are in the vicinity of pH 5 [This IS an exaggeration!]  Where
pH can determine dye binding, pH can be used to partition objects in the
specimen space along a pH gradient [also in Pearse, same pages, but see
Methylene Blue Extinction (MBE) methods in many compendia].  [MBE used to
distinguish among histologic 'acid' mucosubstances (GAG's, etc.).]

	5.  A buffer may stabilize substances, react with them, or promote
changes in them (i.e. oxidation).  

	6.  A bottle of dye, used for 5 years to produce a result, may, at
some moment, STOP behaving as it should.  Pearse mentions that one of the
reasons that metachromasia was such a confused subject prior to the 50-60's,
was due to the fact that so many studies failed to use pure dyes.  

RULE:  If a dye fails to function as it should. Try a different batch or
make up a fresh batch, or purchase a new supply.  I have found that a simple
0.1% solution of the dye gives rise to regular, reproducible metachromasia
which survives dehydration in absolute ethanol but never in 95% ethanol.
That having been said, when I have performed the Azure B, pH 4.0 for
ethanol-acetic acid(3:1)(Clark) fixed nucleic acids (Flax and Himes, 1952),
I follow their protocol for dehydration and use tertiary butanol.
Preparations I made in the mid-60's still show metachromasia, albeit with
some overall loss of color in my personally prepared Damar-xylene mountant.

	The questions about the failure of any regularly used dyeing
procedure amount to a scientific challenge that are best addressed by the
one who is having the problem.  Since the variables are many the sources of
failure are also many.  One has to learn how to perform a component analysis
in order to efficiently address such a problem.  Since much of what one does
in dyeing/staining is by prescribed protocol, it should be clear that if
anything has changed, it is the operator who is in the best position to DO
the troubleshooting.  When I ran the Flax and Himes procedure, I always
retained the blocks of previously sectioned material.   Each sectioned block
was dipped in paraffin to cover the exposed tissue and stored carefully
away.  I was especially careful of those specimens, prepared for any
particular purpose, in the event I ever required a 'known' tissue source of
a good result.  Even though I was aware that the stored specimens would not
be the same in two years as those I sectioned yesterday.  I learned this
when I addressed the issue of saving tissues labeled with tritiated
thymidine.  Why, I asked parenthetically, should I be able to determine that
loss of radioactivity by disintegration was not going to be augmented by
loss due to progressive destruction of DNA, if I had no specific knowledge
of how much DNA/nucleus/section was present in the starting material?  So, I
learned that I would not be able to have compete faith, even in the best of
my archived specimens.

	NOTE:  Acridine orange was one of the first fluorescent dyes used by
virologists in the late 50's/early 60's.  One was able to distinguish
between single stranded RNA and double-stranded DNA until someone noted the,
then recent, discovery of the RNA viruses.  "Nuts!"  Even now, there is an
extensive literature on the use of AO fluorescence for double-dyeing the
nucleic acids in cell nuclei.

	An absolute obligation of old windbags is a SUMMARY:  If there is a
single point in all of this, it is this.  In the application of
metachromasia there are considerations of mass action, pH, purity of the
dye, and treatments preceding and following the application of the dye.  If
each adds an order of magnitude to the number of variables involved, there
are 4-5 such ordinal magnifications through the process.  If the physical
and chemical bases of histologic methods are understood at less than optimal
levels, then troubleshooting will be a problem that has little hope for
success.  On the other hand, even one who has only the recipe to which s/he
can refer can be taught component analysis of that recipe.  

	Mom used to say, "If you don't know what kind of flour to put in
your bread, try any flour and the bread will let you know if you were right
or wrong.  If you were wrong, and really want bread, then you will have to
try another flour, and another, until the bread tells you that you are
finally right.  Just don't change any other part of the recipe while you are
testing the flour."  Ah!  If only more of us had learned to bake when we
were young.

END:  Respectfully submitted,

Fred Monson

Frederick C. Monson, PhD   
Center for Advanced Scientific Imaging
Schmucker II Science Center
West Chester University
South Church Street and Rosedale
West Chester, Pennsylvania, USA, 19383
Phone:  610-738-0437
FAX:  610-738-0437
fmonson@wcupa.edu
CASI URL:  http://darwin.wcupa.edu/casi/
WCUPA URL:  http://www.wcupa.edu/
Visitors URL:  http://www.wcupa.edu/_visitors/




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