PTAH Staining inconsisitant

From:Daniel & Linda Botsford

Any suggestions to correct this problem would be appreciated. We are
getting to much red staining. in muscle tissue. The rbc,nuclei will
stain blue but the muscle tissue remains red when the striations should
be blue. We have tried old reagent vs new reagent. We have tried
increasing the hydration time to the tissue. We are now trying increased
staining time. The tissue in from autopsy. The following is the method.


 This stain is a true stain. During preparation of hemalum solutions,
hematoxylin is oxidized, then the hematein and alum solutions are
combined. Acid is added last after hemalum chelates have been formed. In
contrast, in the traditional phosphotungstic acid hematein (PTAH)
technic, unoxidized hematoxylin is mixed with strongly acid
phosphotungstic acid (PTA) solution. The oxidant potassium permanganate
is then added, removing two hydrogen atoms during oxidation of
hematoxylin. These reactions reoceed smoothly in aqueous or alcoholic
solutions. However, strongly acid solutions contain a large number of
protons that react rapidly with  electron-donating oxygen ion groups of
hematein. Consequently, hematein in acid solution becomes pale to
colourless and shows adsorption spectrum similar to that of hematoxylin,
in which the conjugation (molecular orbitals) of the hematein molecule
has been disrupted. Thus, in traditional PTAH solutions, as permanganate
oxidizes hematoxylin to hematein, the numerous protons of the PTA
solution apparently convert many hematein ions into hemtoxylin-like
products before they can form chelates with tungsten atoms. Therefore,
relatively few chelates are formed during the first few days and weeks.
One day – one week old traditions PTAH  solutions produce moderate blue
colouration of muscle and elastin and pale yellow to orange staining of
collagens. Oxidation of hematoxylin and chelate formation with PTA
continues slowly over several months. Optimal coloration of tissues is
obtained with solutions aged for approximately six months. Such
solutions are then stable for one to three years. From a chemical
viewpoint, it is much more sensible to add the oxidant potassium
permanganate to the aqueous hematoxylin solution,allow the reaction to
proceed for at least 30 minutes, and then mix the hematein and the PTA
solution, following the sequence used in the preparation of other
hematein: metal chelates like hemalum. The huge number of hematein ions
enables PTA ions to compete with protons for electron pairs of –O-
groups to form  chelates. After standing for one hour, the staining
properties of such PTAH solutions are similar to those of traditional
formulas aged for several months.This modified formula is stable for a
year or longer.

 Formation of PT-hematein chelates: The chemistry of chelate formation
of hematein and PTA is in principle similar to reactions with A1+++ or
Fe+++. Hematoxylin must be oxidized to hematein; two hydrogens must be
removed, one from a phenolic-OH group, leaving a phenolate –0- ion, and
one from the central carbon atom. During reaction with a metal atom, the
hydrogen of the –OH group ortho to the –0-  is also displaced. Each –0-
substituent shares an electron pair with the metal, forming a coordinate
bond. Owing to the stearic conditions of PTA red ; 2:1 chelates are
blue. The blue component is sensitive to heat and is partly converted
into red compounds during staining.

 These two fractions are bound to tissues by different chemical
mechanisms. Uptake of the red complex of PTAH is determined by the
preferential reaction of PTA with certain tissue structures, like
collagens. The staining pattern of the red component is similar to those
of PTA-dye combinations in trichrome stains.

 Binding of the blue component of PTAH by elastin, myofibrils  and
fibrin is due to hydrogen bond formation of the two phenolic-OH groups
of each hematein ligand with suiably spaced sites in tissues. As the 2:1
hematein:PTA chelate is rigid, the distribution of hydrogen bonding
sites on tissues must match the spacing of the four phenolic-OH groups
of the chelate. The distibution of the blue component of PTAH is similar
to that of iron hematein  in the modified Heidenhain stain.


 The complete or representative sections of the tissue samples are
received for pathological diagnosis and microscopic examination by the

 Tissue is fixed with 10% NBF, routine processed and cut at 4 microns.


1. Coplin jar

1. Waterbath 56oC

1. 500 ml. beaker

1. 1 liter jar


1.0 Langeron’s Iodine:

This reagent contains Potassium Iodids (KI M.W. 166). It is supplied by
Fisher and is TOXIC (see MSDS). It is stored in the reagent room at room
temperature. Use gloves. This reagent contains Iodine (I M.W. 126.9). It
is supplied by Fisher and is TOXIC (see MSDS). It is stored if the
reagent room at room temperature. Use gloves.
 .1 Potassium Iodide  2 grams
 .2 Iodine   1 gram
 .3 Distilled water 200 mls.

  Transfer to a glass bottle and lable PTAH “Langeron’s Iodine”,
                    date of preparation and an expiration date of one
year. Store at
                    room temperature in special stain box.

1.0 5% Sodium Thiosulphate:

           This reagent contains Sodium Thiosulphate (NaSIO3*5H20
                    M.W. 248.81). It is necessary to weigh out 6.5 grams
of this
                    compound to yield 5 grams of Sodium Thiosulohate. It
                    supplied commercially by BDH. It is stored in the
reagent room
                    at room temperature. Use gloves.

 .1 Sodium Thiosulphate 6.5 grams
 .2 Distilled Water  100 mls.

  Transfer to a glass bottle and label PTAH “5% Sodium Thiosulphate”,
date of preparation, and an expiration date of one year. Store at room
temperature in special stain box. Discard after use.

1.0 Solution “A”:

This reagent contains Hematoxylin C.I.# 75290. It is supplied by Fisher
and is stored in the reagent room at room temperature. This reagent
contains potassium permanganate (KMn04 M.W. 158.03) and is an OXIDIZER
(see MSDS). Itj is supplied commercially by BDH. It is stored in the
reagent room at room temperature. Use gloves.

 .1 Hematoxylin Crystals 1 gram
 .2 Distilled Water  500 mls.
 .3 Place in 56oC waterbath
 .4 Mix until dissolved
 .5 Remove from waterbath
 .6 Wait 10 minutes
 .7 Add .177 potassium permanganate
 .8 Mix until dissolved
 .9 Wait 30 minutes (minimum)

  Transfer to glass bottle and label PTAH “Solution A”, date of
                     preparation. Store at room temperature in the
special stain box.

1.0 Solution “B”:

This reagent contains phosphotungstic acid (CAS 12067-99-1) and it is
stored in the histology refrigerator. It is supplied by Fisher. Use

 .1 Phosphotungstic Acid  20 grams
 .2 Distilled Water  500 mls.

  Transfer to glass bottle and label PTAH “Solution B”, date of
  Preparation. Store at room temperature in the special stain box.

1.0 PTAH Stain:

This is made in-house from Solution A and Solution  B”. Use gloves.

 .1 Solution A  500 mls.
 .2 Solution B  500mls.
 .3 Mix
 .4 Wait one hour

  Transfer to glass bottle and label PTAH “PTAH-Stock”, date c
  Preparation and an expiration date of one year. Store at room
temperature in the special stain box. Do not pour used solution   back
into the stock bottle.

Quality Control

 A quality contol sheet is handed in with the slides to the pathologis
 control slide containing muscle and collagen (skeletal muscle) is run
 with patient slide to ensure the reagents are working.


1.0 Deparaffinize

.1 Remove metal rack with slides from hot air oven and cool 1
.2 Immerse rack in the H&E set-up.
.21 Xylene 4 minutes.
.22 Xylene 4 minutes.
.23 100% Ethanol 2 minutes.
.24 95% Ethanol 2 minutes.
.25 70% Ethanol 2 minutes.
.26 Running tap water 15 minutes or longer.
.27 Distilled Water.

1.0 Staining

.1 Place sides in coplin jar containing Langeron’s Iodine.
.2 Place lid on coplin jar.
.3 Wait 20 minutes.
.4 Place slides in Sodium Thiosulphate.
.5 Wait 1 minute.
.6 Wash slides in running water,
.7 Wait 5 minutes.
.8 Place slides in coplin jar containing PTAH stain.
.9 Place lid on coplin jar.
.10 Place jar in 56oC waterbath.
.11 Incubate for 2 hours.
.12 Remove jar from waterbath.
.13 Dehydrate.
.14 Clear.
.15 Coverslip with resinous mounting media.
.16 Label slide with paper label.
.17 Log into special stain book.
.18 Check control slide microscopically.
.19 Hand slide into pathologist with Q.C. sheet.


The pathologist examines the stain and reports.

Blue – A band of striated muscle, smooth muscle, fibrin, nuclei
Dark Blue – Elastin Fibers
Red – Collagen, reticulum, basement membrane, amyloid


1. The temperature of Solution A must not exceed 60oC.

2. PTAH staining solution is stable for several months. The useful life
 can be greatly extended of portions used for staining are not poured
 back into the stock solution but are kept separately for further use
 until signs of exhaustion become noticeable. If stored in a dark bottle

 away from strong light it may remain good for 18 months to several

3. Thorough washing of sections prior to staining is essential.
 to physico-chemical data, the diffusion rate of solutions is a function

 of flow rate. Dehydration and embedding remove bound water from
 tissues and thus narrow intermolecular spaces. During washing, water
 molecules penetrate and are bound by tissue structures and widen
 internal pathways. Quantitative studies demonstrated increases in the
 diameter of interfibrillar spaces from 5-6 angstroms to 40-100
 angstroms in well hydrated samples. This greatly facilitates uptake of
 dyes especially large molecules like PTAH.

4. Pretreatment of sections with Zenker or potassium permanate  and
oxalic acid is not necessary when using the modified formula.

4. Pretreatment with Langeron’s iodine solution is essential for
binding of PTAH. The chemical mechanism of this effect is not
completely understood. In contrast to the oxidant iodate, iodite is
moderately effective reducing as a reducing agent that can share its
electrons with other compounds.

6. Sodium thiosulphate interferes with the binding of PTAH. Therefore,
sections should be washed in running tap water to remove any trace of
this reagent. Use clean glassware. Staining jars that have been in
contact with sodium thiosulphate should be flushed with copious amounts
of water; even small amounts of reagent will ruin the PTAH solution.


7.0 Most dyes interact in aqueous solutions, forming aggregates that
cannot penetrate dense tissue structures. Therefore textile dryers heat
solutions during drying to break up aggregates and facilate movement of
dyes into fibers. Besides speeding up staining, heating causes a
decrease in the blue and an increase of the red fraction; therefore, do
not use a batch of dye twice on the same day. The blue component is
reconstituted during standing at room temperature for 15-24 hours.

8.0 For optimal staining sections should be put into the PTAH solution
room temperature and then placed into the oven at 56oC. Preheating the
dye batch or heating above 60oC greatly increases the red fraction of
PTAH, affecting staining patterns.

9.0 Over fixation of the peripheral of tissue blocks is called case
hardening. When tissue is placed in fixatives, reagents penetrating
blocks react first with available groups near the surface and cross-link
proteins. Consequently, movement of fixing agents into the central areas
of the blocks is delayed and reactive sites may not be saturated with
reagents. These different degrees of fixation, especilly of dense tissue
like muscle, collagen in skin, and tendon can effect dye binding. The
fixation artifacts described, patchy or predominantly red colouration of
peripheral areas in sections cannot be corrected. Case hardening can
usually be prevented by trimming blocks to 4 mms. Thickness and limiting
the duration of fixation to 16 hours of Zenker-formal or 24-48 hours in
formalin, followed by washing in tap water for 15-24 hours.


Mallory F.B. A Contribution to Staining Methods. III. Phosphotungstic
Acid-Hematoxylin for Neuroglia Fibers. J.Exper Med. 5:19-20, 1900.

Maloan S.N., Puchtler H. On the Chemistry of Phosphotungstic
Acid-Hematein: Development of a Rapidly Ripening PTAH Solution. J.
Histotechnology Vol. 11. 3:153-157, Sept. 1988.

Sheenan, D. Theory and Practice of Histotechnology. 2nd edition.
Toronto, Mosby Company, 1980.                    

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