Re: [Histonet] Russell-Movat's stain - again (LONG)

From:John Kiernan

Dear Nora & all,

I've never done the method, but have looked it up in
Bancroft & Gamble, and also looked at the properties
of the dyes, so here are some facts and suggestions.

The name crocein scarlet is applied to at least 4
similar anionic disazo dyes (CI 26905, CI 27155, CI 27160
CI 27165). They all have various synonyms. Acid fuchsine
is an anionic triphenylmethane dye with molecules about
the same size (MW 550-600 or so), so all these dyes should 
have similar staining properties.

The action of phosphotungstic acid (PTA) is to attach to 
collagen, expelling the red anionic dye(s) from the collagen 
fibres but not from cytoplasm, fibrin etc. The exact 
mechanism by which PTA does this is debatable. 

The sections are rinsed in weak acetic acid rather than
to prevent extraction of the anionic red dyes from stained 
structures. Water counts as slightly alkaline where dye 
anions are concerned. 

In being extracted by acetic acid, your red dyes are
behaving like cationic rather than anionic dyes. One
explanation might be that the triphenylmethane dye is
basic fuchsine rather than acid fuchsine. As a cationic dye,
basic fuchsine is extracted from tissues by acids. If your
crocein scarlet + acid fuchsine solution contains an excess
of basic fuchsine cations over crocein scarlet anions, the
net result will probably be that the sections are being
stained only with basic fuchsine. 

Unfortunately there's a typo in Bancroft & Gamble (p.160)
and the prescribed amount of acid fuchsine for the mixture 
is omitted, so I can't work out the molar amounts 
of crocein scarlet and acid (or basic) fuchsine.

All this is speculation, of course, based on the simple
rule that simple acids extract basic dyes, not acid dyes
stained tissues. 
(There may be some exceptions, expecially among dye-metal 
complexes in stains such as haemalums and
iron-haematoxylins. Also, PTA is not a simple acid like
acetic or HCl; it has complicated reactions and interactions
with dyes of different types.)

Making up a solution with basic fuchsine instead of acid
fuchsine is an easily made mistake because there's quite a
range of names to be found on bottle labels: Fuchsin, acid;
Fuchsin, special for DNA; Fuchsin basic, special for
etc etc. They nearly always omit the terminal e of fuchsine
too. That e is there for a good reason. You won't find
"fuchsin" in American (Webster's) or English (Oxford;

A more serious possibility is that one or both of the
red dyes is not what the label on the bottle says it is.
Does the acid fuchsin(e) bottle carry a Biological Stain
Commission (BSC) certification sticker? It's a little square 
label that includes a reproduction of the late H.J.Conn's
signature. Every batch of a dye certified by the BSC has
several chemical tests and has also been found satisfactory
staining methods that require correctly identified dyes with
adequate dye-content and a low level of coloured impurities. 

Nearly all dye powders contain non-dye ingredients such as 
dextran, NaCl, Na2SO4 etc, and also coloured impurities that
are by-products of the complex chemical reactions that
generate synthetic dyes. 

Some dyes sold as stains are not what they claim to be, 
and they do not work. This was a major problem, especially 
in the USA, in the first half of the 20th century, and it 
is one of the reasons for the existence of the Biological 
Stain Commission.  The BSC has been testing stains since 
1925. In the early 1990s the BSC detected fake dyes 
purporting to be light green SF (used in the Papanicolau 
method for cervical cytology smears). The matter was soon 
resolved, and reported in the BSC's journal, 
Biotechnic & Histochemistry, which is not expensive,
even for an individual subscriber.

I hope this advice helps. If dye identity is a problem,
seek advice from the BSC.

John Kiernan
London, Canada.


Nora Berghoff wrote:
> Hello everyone,
> is there really nobody who is familiar with the Russell's modification
> of Movat's pentachrome and who may have an idea as to what the problem
> may be....?
> Here is the problem (previous e-mail) again, just in case (I am not
> giving up yet):
> ****
> I have a question about Russell's modification of Movat's pentachrome
> stain.
> (Ref.: Russell, HK, "A Modification of Movat's Pentachrome stain", Arch
> Path, Vol. 94, Aug 1972, 187-191)
> I have been staining canine intestinal sections that have been fixed in
> 10% neutral buffered formalin, embedded in paraffin and cut at 5um.
> The stain worked pretty well and gave good results for a while. Last
> week I have had the following problem:
> After destaining the crocein scarlet-acid fuchsin with 5%
> phosphotungstic acid and then rinsing it in 0.5% acetic acid, it kept
> destaining the tissue until everything was just very pale red (one slide
> was completely destained - no red stain left whatsoever and the other
> stains were pale as well).
> I always control the destaining under the microscope and stop when
> there is still intense red color.
> The color kept coming off the slide in the acetic acid solution and
> also in the following rinses in absolute alcohol.
> I make the solutions (phosphotungstic acid and acetic acid) fresh right
> before I need them.
> Does anyone have an idea what the problem might be? Is the acetic acid
> not strong enough/the phosphotungstic acid too strong, etc?? Is it a pH
> problem?
> ****
> As I previously said, I have tried to find an answer somewhere else
> before bothering this e-mail list, but I simply can't find anything. And
> I am afraid I am not experienced enough to detect simple errors that may
> be obvious to someone else...
> So if there is anyone who has even a vague idea of what may cause these
> problems, PLEASE reply...My samples are very valuable, which is why I
> don't want to keep staining (and then discarding) sections
> unnecessarily, just hoping for the problem to resolve on its own.
> Thank you!!!
> Nora
> Nora Berghoff
> Research Assistant
> Texas A&M University
> College Station, TX
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