Staining with hematoxylin and eosin-xylol

Primary Fixation

Fix specimens or tissues and affix them to slides or coverslips. AFA is the preferred fixative. We move our coverslips from columbia jars to ceramic staining racks for processing. All stains and solutions are kept in screw top jars. We remove the lids as needed and move the racks from jar to jar and then tighten down the lids for storage.

Fixative Rinse

Rinse out excess fixative in 70% Ethanol for 3 minutes. Specimens that have been stored in 70% ethanol can go directly to staining.


Harris hematoxylin is water based, so specimens have to be hydrated before staining. Hydrate by graded alcohol series 50%, 3 min; Water, 5 - 7 min.


Harris Hematoxylin is a regressive stain, that is, specimens are overstained and then destained to remove excess stain and differentiate structures. Stain in Harris Hematoxylin for 15 minutes.

Destaining and differentiation

Rinse out excess stain in gently running tap water for 3 minutes and proceed to destaining. Destaining and differentiation is the critical step that determines the quality of the final preparation. Destain in 1% acid alcohol until the cytoplasm has only a faint stain but the sharp nuclear stain still remains. Briefly rinse in tap water to remove excess acid and halt destain.

Bluing and setting hematoxylin

Dip in ammonia water or lithium carbonate water until specimens are bright blue (3-5 dips)and rinse in gently running tap water for 10 - 20 minutes. Hematoxylin is pH sensitive shifting from red under acidic conditions to blue under alkaline conditions. The red state is unstable and fades over time or when exposed to light while the blue state is time and light fast. Thus the bluing step sets the hematoxylin and ensures stain longevity in the final preparation.


Dehydrate in a graded ethanol series: 50%, 70%, 95% (3 min each); 100% (3 changes, 3 min each).

Clearing and counterstaining

Clear in a graded xylene series: 50% xylene/50% Absolute alcohol (3 min); Zelmer's Eosin-Xylol (3 min); 100% xylene (2 changes, 3 min each).


Mount in warm (37-40 C) Damar Balsam. (Note: Canada Balsam tends to become acidic as it polymerizes and will color-shift and fade hematoxylin stains over time.)


Previous Techniques home Next

    Coors coverslip staining rack (left), Columbia coverslip jar (right), coverslip storage box (rear).

    Staining series in screw top jars.

    Hematoxylin and eosin stained gregarine, bright field microscopy.

Harris Hematoxylin

Hematoxylin Crystals
2.5 g
100% Ethanol
25 ml
Ammonium or Potassium Alum
50 g
Distilled Water
500 ml
Mercuric oxide (red)
1.25 g

In separate flasks, dissolve the hematoxylin in the alcohol and the alum in the water with heat. Remove from heat and mix the two solutions. Return to heat and bring to a boil as rapidly as possible (limiting this heat to less than 1 min and stirring often). Remove from heat and add the mercuric oxide slowly while stirring. Reheat to a simmer until solution becomes dark purple (darker than burgundy, lighter than merlot). Remove from heat immediately and plunge the vessel into a basin of cold water until cool. The stain is ready to filter and use as soon as it is cool. Filter before storage. Before use, add 2 - 4 ml of glacial acetic acid per 100 ml solution to increase precision as a nuclear stain. Shelf stable for about a year.

Zelmer's Eosin-Xylol

Eosin Y, water soluble
1 g
Distilled water
100 ml
HCl, concentrated
2.5 ml
100 ml

Dissolve the eosin in the water. Add the hydrochloric acid and mix well. Allow the precipitate to settle overnight and decant the clear supernatant. Add Xylol to the precipitate and shake gently or stir with a glass rod, avoiding an emulsion. During this process the free acid of eosin is dissolved in the xylene layer. Allow to form an organic - inorganic interface and decant the nearly colorless xylol extract which stains organic tissues on contact. Shelf stable. Can be diluted 1:1 with xylene for counterstaining.

1% Acid Alcohol Destain

1% vol/vol HCl in 70% ethanol.

5% Acid Alcohol Destain

5% vol/vol HCl in 70% ethanol.

Ammonia water

Mix 2-3 ml Ammonium hydroxide (28%) per liter of water. Stores for about a weekly.