Hi All,
Mordanted hematoxylin is quite colorfast when carefully 'blued' after binding
to acid moieties in the tissues. If one analyzes the entire process of H&E
dyeing, one can 'see' several steps in which the 'bluing' process can be
'undone' or 'temporized'. Note that I have always used personally prepared Gum
Damar in Xylene to mount my sections. It has always behaved better than
expected.
Please recall that all of the currently used histologic procedures originated
using water glasses from the dining room or kitchen and chemicals that were
generated by magic. When I was learning the procedure using the now-antiquated
Coplin jar, I was overwhelmed by the inability of distilled water to 'blue'
hematoxylin after acid differentiation. For we new histologists at the time,
the only thing that really 'set' the blue color was running tap water for 5
minutes AFTER 5 minutes in 35% EtOH, 2% in NaHCO3. NB, the same magic rinse
was required to stabilize the magenta of the PAS stain. In those days, there
was the complaint that even some tap waters in other parts of the country left
some lack of color fastness with mordanted hematoxylin (it must be remembered
that the active component of the H&E stain is NOT hematoxylin (those who are
surprised must go the library!!)
In any case, I had a colleague who noticed that the slides in his collection
were fading slowly in the closet in which he stored them (stored pretty much as
we all do). It was suggested that he perform a test of the storage environment
in the following way.
Using an aquarium aerator placed in the closet to bubble air thru 1.5
liters of distilled water with an appropriate indicator in a two-liter
Ehrlenmyer with a two-hole stopper, the outlet taking the bubbled air out of
the closet via an appropriate length of tubing. Modify the above aerator and
water volume to insure that the water will not be significantly evaporated
during the overnight process (temperature, volume, and pressure are the
appropriate variables).
This test should be run for several to many nights and days (or weeks) to
determine whether at any time the environment 'goes' acidic. [[The upside is
that the experiment/test is neither labor intensive nor expensive (no grant
proposal is required), though if you can wangle some stimulus money, you must
let me know, because they will want to be able to count me as one of those who
was put to work - albeit briefly. If you run the above test for several years,
you may be able to count me as being employed every time you refer to this
suggestion. [Perhaps not ethical, but we must do anything to keep unemployment
under 10% lest there be declared another economic crisis whose fix we know by
now will be the expected printing of more money to fund more studies of more
fish in a pond or bugs on a wall.]]
Back to point, if at any time one observed the water to go acid, then the
location of the storage is a likely candidate.
If you use a machine to stain your sections, as many to most do, then perhaps
another environmental characteristic (such as humidity) is causing either
continuous or seasonal ethanol dilution and thus leaving too much water in the
sections, even though they do not appear milky when mounted. In such a case,
one must expect faster diffusion of acidic materials into the preparation. NB,
I only consider those airborne substances that are hydrogen rich - they can be
inorganic or organic - simply anything that gets sprayed, mixed, dumped in the
sink - anything that continuously gets aerosoled in the lab or is blown in with
the 'conditioned' air.
Another test you might perform involves sealing slide boxes in plastic bags -
see your local molecular biologist or check the kitchen area at KMart, Penny's,
or Walmart for systems that pull a vacuum around food and seal it by creating a
heat seal. [If still unfamiliar, call Mom OR Mom-in-law!]. You must have a
control box to compare over time. One assumes that NOTHING can get in or out
of such a sealed container.
The ultimate downside is that one does not or cannot control a process that one
trusts to be automatic and credible.
Hematologists may take note: At the age of 40 I was laid up with a pulmonary
'problem' that included a few days with 104-105 degree spikes, dry coughing,
and visits from the Savior who I sent away screaming, "Hell No! I won't go!" -
and in the process scarring the b'Jesus out of my kids. In any case after
passing the hospitalization test and ahaving received, i.v., every known broad
spectrum antibiotic in the pharmacy, I got my doctors to give me erythromysin
i.v.("we don't give enythromysin orally or i.v. - it bites!), the temp went
down, and I felt better. [If legionella is sensitive to erythromysin, why did
so many die from that bug in Philadelphia? (This will not be on the test, but
it should be.)] Thus, came the day when I was told that my blood differential
was so messy that it had been recommended that I submit to a bone marrow exam
to determine my proximity to death. I quietly demanded to know how the
differential was done. The answer? The Coulter counter! I quietly agreed to
a bone marrow exam AFTER a manual differential was performed. Later, the
hematologist only wanted to know how I knew. I told her that while working in
a path lab at night during college (I know I wasn't 'certified'!!!), I had
compared myself to the 'Counter' and I was frankly more consistent. I admitted
that my test was not statistical, but I had real confidence in the capacity of
the eye and little in the photometer looking thru glass.
Machines may be necessary to do the work, but they do not clean themselves and
they do not cry out when the 95% ethanol has become 87%. With Coplin jars,
variables were easy to manage. They were more difficult the more we tried to
do multiples of specimens. This should be obvious in spite of the fact that
automation is practical and manual is not - even though sometimes necessary.
When I started in histology, I began at the beginning of the Paraplast
revolution, yet there were still those who formulated their own paraffin for
the part of the planet in which they worked.
Sorry for the late-night babble, but I have vented stored up compulsion.
Cheers,
Fred Monson
P.S.
Those who are required to volunteer do NOT. Those who require others to
volunteer fail.
Every tax is a means by which a government forces a citizen to pay for the
vacations of those who work for the government.
When the USA has a public health plan, every union will be forced to accept it
by the employer, and all those who want something better will be required to
pay for the full load. Government plan - 'free', all others - cost extra.
That's competition? Why won't the insurance companies want to give 'free'
public health care to their employees? Their own actuaries will show them the
way.
Three of four wars prosecuted by the United States during the 20th century were
begun under democratically elected Presidents.
One was fought entirely by volunteers.
During only two of the four have a majority of citizens, at one time or
another, wished the U.S.A. to lose(???).
Three cheers for the Draft!!!!!
Beware the resolve of an elected President not to lose.
Frederick C. Monson, PhD
Technical Director, CMIRT
West Chester University
West Chester, PA, 19383
610-738-0437
http://cmirt.wcupa.edu
-----Original Message-----
From: histonet-boun...@lists.utsouthwestern.edu on behalf of Rene J Buesa
Sent: Thu 7/2/2009 3:27 PM
To: histonet@lists.utsouthwestern.edu; sr...@aol.com
Subject: Re: [Histonet] Help With Hemo Fading
The fading most probably is caused by acid in the permanent slide, probably
because the sections were passed through the alcohols very quickly after the
acid differentiation, or they stayed little time in tap water after
differentiation or no bluing agent was used.
It is unlikely that the mounting medium is acidic, although that could also be
the cause also. An acid environment over the cover slipped section is the most
probable "culprit" for the henatoxylin fading.
Check the staining protocol.
René J.
--- On Thu, 7/2/09, sr...@aol.com <sr...@aol.com> wrote:
From: sr...@aol.com <sr...@aol.com>
Subject: [Histonet] Help With Hemo Fading
To: histonet@lists.utsouthwestern.edu
Date: Thursday, July 2, 2009, 2:42 PM
Hello everyone,
?
We are having problems with short-term hematoxylin fading and loss of detail.
The pathologist is freaking out! I've seen hemo fade over a long period of time
but not in a matter of a few months. Slides from one year ago are really bad.
?
I've been out of the business for a number of years and in the interim much has
changed including reagents. These are GI tract biopsies processed by microwave.
?
Any thoughts at all?
?
Thanks! Marg
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