HAEMOTOXYLIN

It is natural dye derived by the extraction of the wood of a Mexican tree Haemotoxylon

Campechianum. Haemotoxylin has poor staining properties and is normally used in

conjunction with a mordant. If the staining solution is oxidized, the staining intensity.  The first way of doing ripening is spontaneous or natural. The staining mixture is

placed in air openly and atmospheric oxygen brings ripening. It is very lengthy process and

takes 3 ± 4 months.

Other way of ripening is by chemical oxidants, i.e. some chemical agents are used for

ripening e.g. mercuric oxide. An exact amount of chemical oxidant is used with respect to

the amount of haemotoxylin. When the oxidant is in excess, over ripening result. The exact

quantities of oxidants are:

HX OXIDANT

1g 0.5 g(HgO)

1g 0.175 g (KMnO4)

1g 0.05 g (KIO4)

1g 2mL (H2O2)

Mordant is substance which interlinks dye molecules and tissue components. Haemotoxylin

is called indirect stain because it requires mordant. The mordant is indispensible for staining

reaction. In haemotoxylin staining, divalent or trivalent metallic salts are used, although

simple salts of metals like aluminum (Al), Iron (Fe) Chromium (Cr) can also be used but

conventionally alum salts are used in this staining.

K2SO4.Al2 (SO4)3.24H2O (Potassium Alum)

(NH4)2 SO4. Al2 (SO4)3.24H2O (Ammonium Alum)

(NH4)2 SO4. Fe2 (SO4)3.24H2O (Iron Alum)

Simple chloride and sulphate salts of aluminium can also be used. Use of alum slats is just a

tradition because initially these were cheaper.

Preparation

In routine, Harris haemotoxylin is used. In this type ripening is done by mercuric oxide

(HgO) potassium and aluminium is used as a mordant.

Ingredients

Hx Stain Powder 2.5 g

Absolute Alcohol 25 ml

Potassium Alum 50 g

Distilled H2O 500 ml

Mercuric Oxide 1.25 g

Glacial Acetic Acid 20 ml

Significance of Ingredients

R Hx is a stain /dye.

R Absolute alcohol is solvent for Hx powder. 

R Alum is mordant.

R Dist. H2O is solvent for alum. 

R Mercuric oxide brings ripening process.

R Glacial acetic acid enhances staining intensity. 

Procedure

1. Dissolve Hx powder in absolute alcohol and alum in distilled H2O with gentle heat.

2. Mix both solutions and bring to boil rapidly. Then remove container from flame and

add HgO. 

3. Cool the mixture rapidly and add glacial acetic acid.

4. Filter the stain and store in dark coloured bottle.

EOSIN

Eosin is commonly used as a countersstain along with haemotoxylin. Eosin is a synthetic

dye and belongs to xanthenes dyes group of stain. Eosin is a type of acidic dye so

cytoplasm, muscle fibers, RBCs and connective tissue fibers etc show affinity for eosin.

Eosin has an excellent property of staining different tissues components in different shades

ranging between orange to deep red. Different brands of eosin are available, eosin Y, B, S.

Eosin Y is commonly used. Orange G phloxine is a substitute.

Preparation

1. Eosin powder 1g Dist H2O 100 ml

2. K2 Cr2 O7 1g Dist H2O 100 ml

Prepare separately and then mix both solutions. 

H/E Staining Procedure

Reagents Time Rationale

Xylene I 5-10 min De-waxing or

Xylene II 5-10 min Deparaffinization

Abs. Alcohol I 2-3 min Dexylenation

Abs. Alcohol II 1-2 min

90% Alcohol 1-2 min

70% Alcohol 1-2 min Hydration

50% Alcohol 1-2 min

Running tap water 1-2 min

Hx Stain 1-2 min

Progressive

Nuclear Staining

3-5 min

Regressive

1% Acid water

(only for

regressive)

1-2 dips Differentiation

Alkaline

Running tap water

2-3 min Bluing

or (1% lithium

carbonate)

Eosin Stain 3-5 min Cytoplasmic Staining

Running tap water 1-2 min Excess Stain Removal

70% Alcohol 1-2 min

90% Alcohol 1-2 min Dehydration

Abs. Alcohol 1-2 min

Xylene I 3-5 min Clearing

Xylene II 3-5 min

Mounting of sections by a mounting media (Canada Balsom).

Results

Nuclei Blue

Cytoplasm Pinkish Red

Muscle fibers, Fibrin Deep Red

Collagen Fibers Pink

RBCs Orange

Bluing

In Hx staining dissociation of Al2 (SO4)3 takes place in aluminium ions and sulfate ions

whereas some molecules of H2O dissociate into OH- and H+ ions.

Al2 (SO4)3 Æ 2Al3+ + 3SO4

-2

H2O ÆÅ H+ + OHAluminium ions combine with OH ions to form insoluble aluminium hydroxide which

combines with dye molecules to form a complex

with tissue components to bring about Hx staining. There are different sources of H+ ions in

Hx solution like dissociation of water, dissociations of acetic acid and H+ ions from

differentiators. When H+ ion are in excess, they suppress the concentration of OH- ions

which are then not available for the formation of Al (OH)3 necessary for Hx staining. In

bluing, excess of H+ ions are neutralized by some alkaline medium to make OH- ions

available, for Al (OH)3 formation.

Thus bluing is a neutralization reaction because Hx solutions having H+ ions in excess are

reddish and when H+ neutralized, Hx solution turns blue from red. The alkaline medium can

be running tap water (alkaline), 1% lithium carbonate or 1% ammonia water.

Differentiation

Nuclei show affinity for basic dyes and cytoplasm for acidic dyes but these affinities are not

absolute, i.e. certain part of the nuclei are stained with acidic dyes and so is in the case with

cytoplasmic parts which are stained with basic dyes. In Hx staining relative removal of the

dye is performed which means it is removed from cytoplasmic parts while nuclei still

remain strongly stained with Hx stain. This selective removal is known as differentiation. It

is performed in 1% acid water or 1% acid alcohol. The staining is known is Regressive

Method of staining.

Sometimes differentiation is not required and Hx staining is controlled by reducing this

time. This way of staining is called Progressive Method of staining. So Hx can be used

regressively or progressively.

METACHROMATIC STAINING

Metachromasia is a property by which a certain tissue component is stained by a certain dye

in shade or colour totally different from the original colour of the staining solution. The

stains which stain the tissue components in different colour than their original colour are

. The metachromatic property depends on:

x Nature of Dye

x Nature of Tissue Component

Nature of Dye:

All metachromatic stains are basic in nature and contain large cationic Radicals. 

Polymerization. Their metachromatic property is totally dependent on polymerization. For

example, original solution of toluidine blue comprises of monomeric units and is blue in

colour. When it is applied to tissue, dimers and trimers are initiated to form due to which

violet colour is produced. As polymerization is completed, a complete metachromatic

colour appears, i.e. Red. 

Nature of Tissue Component:

About tissue components it is found that these are composed of large anionic radicals like

phosphates (PO4), sulphates (SO4) carboxylic groups etc. These are acidic in nature.

Formerly such tissues components were called Acid polysaccharides. However, now a new

name Glycosaminoglycans is given to these tissue components. Example of such

components are heparan sulphate (heparin) concentrated in mast cell granules, chondroitin

sulphate richly present in cartilage matrix and sometimes dermatan sulphate and keratan

sulphate etc.

In all these substances, anionic radicals are very close to each other (0.5 nm apart) and

when stain is applied to such tissue components, basic cationic radicals are primarily linked

with acidic anionic radicals to form salts. At the same time a secondary linkage between the

dye molecules take place which is due to the proximity of anionic radicals of the tissue

component and facilitate the polymerization. Glycosaminoglycan joins with protein to form

proteoglycans, so proteoglycans and sulphated glycosaminoglycan both show

metachromatic property because these are Acid Mucopolysacharide.