There are many varieties and uses of hematoxylin.  The perhaps best well known is the use of the hematoxylin and Eosin (H&E) staining procedure.

For those who do not already know, the H&E is a routine satin done as the first stain on all projects to understand the basic morphology of the tissue you’re looking at.  The hematoxylin in this stain is used to find the nuclei of all types of tissues.

Hematoxylin can be used in two different methods.  There is the Progressive method, where the staining is done by creating a hematoxylin solution which has an excess of salts or acid.  This increases the selectivity for nuclei.  The regressive staining involves over staining of the tissues in a relatively neutral hematoxylin.  The slides are then brought through an acid wash, which takes off excess stain followed by an base wash to neutralize the the acid process.  This is more difficult than it looks and takes practice.

The hematoxylin recipes here are most of the most common ones used although most labs tend to buy pre-made kits.   Here are the 6 recipes in type and PDF.

Harris Hematoxylin

Harris hematoxylin – 5.0 gm

Alcohol, 100% – 50.0 ml

Ammonium or potassium alum – 100.0 gm

DH2O – 1000.0 ml

Mercuric oxide – 2.5 gm

Dissolve the hematoxylin in the alcohol, the alum in the water by the aid of heat.  Remove from heat and mix the two solutions.  Bring to a boil as rapidly as possible.  (Limit this heat to less than 1 minute and stir often). Remove from heat and add the mercuric oxide slowly.  Reheat to a simmer until it becomes dark purple, remove from heat immediately and plunge the vessel into a basin of cold water until cool.  The stain is ready for use as soon as it cools.  Addition of 2-4 ml of glacial acetic acid per 100 ml of solution increases the precision of the nuclear stain.  Filter before use.

Mayer’s Hematoxylin

Hematoxylin crystals – 1.0 gm

DH2O – 1000.0 ml

Sodium Iodate – 0.2 gm

Ammonium or potassium alum – 50.0 gm

Citric Acid – 1.0 gm

Chloral hydrate – 50.0 gm

Dissolve the aluminum in water, without heat; add and dissolve the hematoxylin in this solution.  Then add the sodium iodate, citric acid and the chloral hydrate, shake until all components are in complete solution.  The last color of the stain is reddish-violet.  Stain keeps well for months.

Delafield’s Hematoxylin

Hematoxylin crystals – 8.0 gm

Alcohol, 95% – 50.0 ml

Ammonium or potassium alum, saturated aqueous solution

(approx. 15 gm/100ml)

Add the hematoxylin dissolved in the alcohol to the alum solution and expose to the light and air in an unstoppered bottle for 3-5 days.  Filter and add:

Glycerin – 200.0 ml

Alcohol, 95% – 200.0 ml

Allow the solution to stand in the light about 3 days, filter and keep in a tightly stoppered bottle.

Bohmer’s Hematoxylin

Hematoxylin crystals – 5.0 gm

Ammonium or potassium alum – 100.0 gm

Alcohol, 80 % – 60.0 ml

DH2O – 1000.0 ml

Dissolve the hematoxylin in the alcohol and the alum in DH2O.  Mix and ripen by exposure to light and air for about 2 weeks.

Bullard’s Hematoxylin

Hematoxylin crystals – 8.0 gm

Alcohol, 80 % – 144.0 gm

Glacial Acetic Acid – 16.0 ml

Dissolve by gentle heat and add mixture of:

Ammonium or potassium alum – 20.0 gm

DH2O – 250.0 ml

Heat to boiling and then remove from flame and add slowly.

Mercuric Oxide – 8.0 gm

Cool quickly, filter and add mixture of:

Alcohol, 95% – 275.0 ml

Glycerin – 330.0 ml

Glacial Acetic Acid – 18.0 ml

Ammonium or potassium alum – 40.0 gm

Ehrlich’s Hematoxylin

Hematoxylin crystals – 4.0 gm

Alcohol, 95% – 200.0 ml

Ammonium or potassium alum – 6.0 gm

DH2O – 200.0 ml

Glycerin – 200.0 ml

Glacial Acetic Acid – 20.0 ml

Dissolve the hematoxylin in the alcohol and the alum in DH2O and mix.  After these are in complete solution add the glycerin and acetic acid.

Note: Aluminum ammonium sulfate is known as ammonium alum. Aluminum potassium sulfate is known as potassium alum. 

Weigerts Iron Hematoxylin solution 

Equal parts of solution A and B

Solution A

Hematoxylin crystals – 1.0 gm

95% Alcohol – 100.0 ml

Solution B

Ferric Chloride, 29% aqueous – 4.0 ml

DH2O – 95.0 ml

Hydrochloric acid, concentrated                – 1.0ml

Ferric chloride 29%

Ferric chloride – 29.0 ml

DH2O – 100.0 ml

Hematoxylin varieties