Hematoxylin Staining

The word hematoxylin is derived from the old Greek words Haimato (blood)
and Xylon(wood), referring to its dark red color in the natural state, and to its method
of manufacture from wood.
Hematoxylin (C.I. 75290) is a natural dye extracted by boiling the wood of the South
American and West Indian logwood tree (Haematoxylon campechianum),
and partly purified by recrystallization. It is sold commercially as a crude mixture of
hematoxylin and other, unidentified substances. It comes as a brownish tan powder
which is poorly soluble in water and somewhat more soluble in ethyl alcohol (1
gm/100ml water and 30-40 gm/100 ml alcohol). The active dye is not hematoxylin
itself, but its oxidized product, hematein. The latter can be bought, but it is expensive,
and the same results can be had simply by oxidizing the crude hematoxylin.
In the early days, oxidation was carried out by making up the hematoxylin solution,
plugging the container with cotton, and leaving it exposed to light and air for 6 weeks
to several months.
However, it was soon found that oxidation (also called ripening) could be achieved
much faster by adding a wide variety of oxidizing agents, the two most common being
sodium iodate and mercuric oxide. With either agent, oxidation not only begins
immediately, but apparently continues for some time, causing dye precipitates of
uncertain composition, and eventually exhausting the stain.
The addition of glycerin to several formulas is said to guard against over-oxidation
and perhaps to retard fungal growth.

Hematoxylinlakes
Used alone, hematoxylin is a poor stain, but in combination with various metallic salts
(mordants) which couple it to the tissue, it is one of the best nuclear stains known. It
has also been used to detect metals in tissues, to stain myelin, to stain mitotic figures,
fibrin, muscle cross striations and other tissue elements.
The combination of hematoxylin plus mordant is called a hematoxylin lake, and lakes
with different metals have different colors. The aluminum lake formed with
ammonium alum (aluminum ammonium sulfate) is particularly useful for staining
nuclei. It is purple in acid solution, but blue in alkaline solution. Instead of aluminum
ammonium sulphate, either aluminum potassium sulphate (potassium alum) or
aluminum sodium sulphate (sodium alum) may be used to create the aluminum lake.

No appreciable difference in results is found. Hematoxylin recipes using any of these

mordants are loosely called alum hematoxylins, orhemalums.
Other hematoxylin lakes are used for different staining purposes, and have a variety of
colors, depending on the metal used.
Metal

Colour

Aluminum

Purple to blue

Iron

Blue-black

Chromium

Blue-black

Copper

Blue-green to purple

Nickel

Violet shades

Tin

Red

Lead

Dark brown

Osmium

Green brown

Numerous formulas for alum hematoxylin have been devised ever since Boehmer first
described it in 1865. Some of the more common are listed elsewhere in StainsFile.
The staining of nuclei by alum hematoxylin is enhanced by the addition of acetic acid,
which apparently reacts with the nuclear chromatin, giving it a somewhat crisper
appearance. However, because of the alum content, the pH of alum hematoxylins
tends to be around 2.62.9 even before the acid is added.

Progressive
vs
All alum hematoxylins, whatever
either progressive orregressive stains.

regressive
their formula

may

be

staining
used as

Progressive staining means that the tissue is left in the stain just long enough to reach
the proper endpoint. Therefore it may be necessary to examine the slides at several
different intervals to determine when staining is dark enough but not too dark.
Regressive staining means that the tissue is deliberately overstained and then
destained (differentiated) until the proper endpoint is reached.

The difference between methods is largely one of convenience. Progressive

hematoxylins are generally less concentrated and work slowly to avoid overshooting
the endpoint.
Regressive hematoxylins are more concentrated and many can achieve overstaining in
a matter of less than a minute, while differentiation (removal of excess stain) requires
only a few seconds. Also, timing is not so important in regressive procedures. As long
as the slide is overstained, it doesn't matter whether it was in the staining dish for 1
minute or 10.
Regressive procedures are therefore faster and more convenient than the progressive
ones, and they have the added advantage that differentiation also removes
hematoxylin from the gelatin or other slide adhesive, producing a clear, transparent
background.
Differentiation is done in dilute acid (usually acid alcohol because of hematoxylin's
greater solubility in alcohol). Differentiation is stopped immediately by simply
washing the slides in water.
If too much hematoxylin has been removed, a few more seconds in the stain will
correct the problem, and the whole process can be repeated.
If too little has been removed, a few more dips in acid alcohol will produce the correct
endpoint.
Note: With all hematoxylins, progressive or regressive, the endpoint is the same!
In a properly stained slide, only the nuclei, flecks of calcium, and certain kinds of
mucus will be stained, while the background will be unstained or very pale brown.
There should be no hint of blue or gray.

Blueing
Because most alum hematoxylin formulae are fairly acid, the nuclei will at first be
stained the purplish color of the acid dye. Changing their color to blue gives
a much better contrast with the usual red counterstains.
When the endpoint has been reached by either progressive or regressive methods,
nuclear color can be changed in one of two ways.

1. The slides may be dipped for a few seconds into a weakly alkaline solution
such as ammonia water or dilute sodium carbonate. Note that differentiation
stopped the second you rinsed the slide in water, so the alkaline solution
is not necessary for that.
2. They may be washed for 2-5 minutes in tap water. Depending on the
geographic area and the local method of water treatment, tap water tends to be
slightly acid, with a pH in the range of 6.0 - 6.8. However, this is considerably
more alkaline than the pH of most alum hematoxylins (2.6 - 2.9), so bluing
results. The tap water wash has the added advantage that it washes out any
excess alum, giving a crisper nuclear stain and preventing fading during
storage.

Variability of hematoxylin
1. The strength of all hematoxylins varies from day to day, becoming a little
weaker with every use because the slide racks carry a little water over with
them, causing some degree of dilution. Also the strength becomes stronger each
time a new solution is made.
2. Oxidation continues slowly and irregularly from day to day, further reducing
the dye strength by producing unpredictable amounts of precipitated dye which
must be filtered out.
3. Oxidation produces several oxidized derivatives of hematein, from
monoxyhematein to pentoxyhematein, each with a different color. The di- and
tri-oxy derivatives appear to offer the optimum color. Tetroxyhematein is
brownish and pentoxyhematein is colorless, so obviously the oxidizing step can
be carried too far and render the dye unusable.
4. To slow down aerobic oxidation of the stock solution in storage, some people
cover the surface with a layer of oil and pipette from below the surface when
more stain is needed.
5. Remember that the original hematoxylin was a crude extract of logwood, so it
may contain hundreds of unknown substances besides hematoxylin, and some
of them and their oxidized products may be responsible for some of the
precipitates.

6. Note the "dye content" on the label of the jar containing the hematoxylin
powder. A dye content of 50% means that only half of the powder is
hematoxylin, and the other half is a mixture of unknown composition.
7. The precipitates may cause obstruction in some mechanical stainers if the dye
must pass through tubes or orifices of small caliber.