Hematoxylin-eosin Staining: Characteristics, Uses, Techniques

The hematoxylin – eosin is a staining technique using the combination of the dyes hematoxylin and eosin. This pair of dyes make a perfect duo, as hematoxylin acts as a basic dye and eosin is an acid dye.

The designation of basic or acid dyes does not refer to the pH they obtain in solution, but rather speaks of the prevailing proportion in terms of the anionic or cationic charges they possess or by the location of the chromophore group.

Hematoxylin-eosin (HE) stained ciliated columnar epithelium Source: Todd Straus and Vladimir Osipov [CC BY 3.0 (https://creativecommons.org/licenses/by/3.0)]

In this sense, hematoxylin is considered a basic (cationic) dye and therefore has an affinity for acid structures, such as the nucleus of cells. While eosin, being an acid (anionic) dye, has an affinity for alkaline or basic structures, such as cell cytoplasm.

For this reason, this combination of dyes is widely used for tissue staining, as it allows nuclei and cytoplasms to be clearly distinguished. The nuclei stain dark blue or purple and the cytoplasm pink.

Hematoxylin-eosin staining is one of the most widely used staining techniques in the area of ​​histology and cytology, due to its easy handling and low cost. 
It is used to visualize cells, thick nerve fibers and the presence of certain microorganisms in tissues, such as: parasites, fungi and bacteria, among others.

Article index

  • one

    Characteristics

    • 1.1

      Hematoxylin

    • 1.2

      Eosin

  • two

    Applications

    • 2.1

      Nerve fiber staining

    • 2.2

      Histological skin section stains

    • 23

      Hematoxylin-eosin staining of stool samples

    • 2.4

      Staining of histological sections for the diagnosis of infection

  • 3

    Techniques

    • 3.1

      For histological samples

    • 3.2

      For stool samples for E. histolytica

  • 4

    Preparation of reagents

    • 4.1

      – Hematoxylin

    • 4.2

      – Eosin

  • 5

    References

Characteristics

Hematoxylin

Hematoxylin is a neutral dye. However, the component that provides the color (chromophore) is located in the cationic or basic center of the molecule. Hence its affinity for acid structures. Its chemical formula is C 16 H 14 O 6 and its scientific name is 7,11b-dihydroindeno [ 2,1- c ] chromene-3, 4,6a, 9,10 (6 H ) -pentol.

It mainly stains the nuclei of cells, since they are very rich in nucleic acids. It can also stain cytoplasmic inclusions of viral origin.

In order for hematoxylin to stain, it must be in an oxidized state and bound to a metal. The latter will serve to fixate to the tissue, that is, it will act as a mordant.

When hematoxylin is oxidized it is called hematein. Oxidation is achieved by exposure to oxygen (aging) of the reagent or by substances that help its oxidation (chemical oxidation).

Eosin

Eosin is a dye that stains red or pink. It is insoluble in water although there is a water soluble version. Generally, eosin is prepared by dissolving in alcohol (95 ° ethanol).

It stains cytoplasms, muscle fibers, cytoplasmic organelles, and collagen, but does not stain cell nuclei. This is because it is negatively charged, therefore, it has an affinity for positively charged structures.

There are two types of eosin “Y” and “B”. Eosin “Y” is known as yellow eosin. Its scientific name is tetrabromo fl uorescein and its chemical formula is C 20 H 8 Br 4 O 5 .

On the other hand, eosin “B” is sometimes referred to as bluish erythrosine B. Its scientific name is dibromodinitro fl uorescein and the formula is C 20 H 8 Br 2 N 2 O 9 . Both are very similar and the difference between using one or the other is not really noticeable. However, the most popular is eosin “Y”.

Eosin has the property of distinguishing between a living and a dead cell, since it is only capable of crossing the membrane to stain its cytoplasm when cells are dead, leaving the cytoplasm of the cell colorless if it remains alive.

Applications

Nerve fiber staining

Thick nerve fibers can be stained and identified with hematoxylin-eosin. However, it is not useful for staining thin nerve fibers, since a silver staining is required to be able to visualize the latter.

Histological skin section stains

In the staining of the horny layer of the skin, the dye that acts is eosin, since at this level the cells do not have a nucleus.

In the granular layer of the skin, hematoxylin strongly stains the keratohyalin granules inside the granule cells. On the contrary, the spinous layer of the skin is weakly stained with hematoxylin, while the basal or germinal layer is sufficiently stained.

Eosin stains the cytoplasm of all cells and the intensity of the color can vary from one layer to another.

Hematoxylin-eosin staining of stool samples

Gómez et al., In 2005 demonstrated that hematoxylin-eosin staining was more effective in identifying cases of amoebiasis due to Entamoeba histolytica and  Entamoeba dispar than the fresh visualization method (saline and lugol) in patients with acute diarrheal disease.

It has also been shown to be highly sensitive in detecting erythrophagocytosis (amoebae that have engulfed erythrocytes).

Staining of histological sections for the diagnosis of infection

Walwyn et al., In 2004 proposed the use of histological stains to detect infection-causing microorganisms.

Using hematoxylin-eosin staining, they were able to visualize infections caused by Clostridium , Actinomyces , spirillae or Candida . They also managed to observe the presence of the parasite Sarcoptes escabiei  in skin sections and viral inclusions by cytomegalovirus and herpes in sections of various tissues.

Techniques

For histological samples

Histological section staining goes through a series of steps. The first thing is to obtain the histological section. This must be waxed to later obtain the cuts (ultra-fine) with a microtome. The technique consists of the following steps:

1-Elimination of excess paraffin : for this you can use xylol or Heme-D, immerse for 3-5 minutes.

2-Rehydration of the sample: this is achieved by submerging the sample in different concentrations of alcohols (ethanol) in descending order (100 °, 90 °, 70 °). In all cases for 7 minutes.

3-Elimination of excess alcohol: for this it is immersed in water for 7 minutes.

4-Staining with hematoxylin: the sample is immersed for 6-10 minutes in a tray containing hematoxylin. The exposure time depends on the size and thickness of the sample.

5-Elimination of excess hematoxylin: it  is washed with water for 5 minutes and then a rapid passage (10-20 seconds) is carried out through acid alcohol. Later it is washed with water again for 5 minutes. Then it is immersed in ethanol at 96 ° for 1 minute.

6-Eosin staining:  for this, the sample is immersed for 5 minutes in the eosin tray.

7-Dehydration of the sample : for this, it is passed through the alcohol trays (ethanol) again, but this time in ascending order. (70 °, 90 °, 100 °). (For 5 seconds, 5 seconds, 1 minute respectively).

8-Clarification of the sample : for this, it is exposed to xylol for 5-10 minutes and dried to seal permanently with Canada balsam or other similar material.

For stool samples for E. histolytica

A smear is made on a slide with the patient’s stool and fixed with 80% alcohol for 5 minutes. The sheet is immersed in hematoxylin for 5 minutes and immediately washed with water.

Subsequently, it is quickly immersed in acidic alcohol and then in ammonia water. It is washed with water. It is colored for 5 minutes in eosin. The sample is dehydrated as explained in the prior art and finally rinsed with xylene.

Preparation of reagents

– Hematoxylin

In one liter of distilled water dissolve 50 grams of potassium or ammonium aluminum sulfate. When completely dissolved, add 1 gram of crystallized hematoxylin. When completely dissolving, 1 g of citric acid is added together with 50 g of chloral hydrate and 0.2 g of sodium iodate.

The mixture is boiled for 5 minutes, then it is allowed to cool and it is filtered to remove any solid particles that have remained. The reagent thus prepared can be used immediately.

– Eosin

It can be prepared with an alcoholic base or with a water base.

Alcoholic eosin

In 100 ml of ethanol at 95 ° dissolve 0.5 grams of eosin “Y”. Then add a few drops of glacial acetic acid.

2% aqueous eosin

In 1250 ml of distilled water dissolve 25 grams of water-soluble eosin “Y”. Then add a few drops of glacial acetic acid.

Acid alcohol

Measure 0.5 ml of concentrated hydrochloric acid and make up to 100 ml with absolute alcohol.

Ammonia water

Measure 0.5 mL of concentrated ammonia and make up to 100 mL with distilled water.

References

  1. Navarrete, G. Histology of the skin. Rev Fac Med UNAM 2003; 46 (4): 130-133. Available at: medigraphic.com
  2. Gómez-Rivera N, Molina A, García M, Castillo J, Castillo J, García R, Fonseca I, Valenzuela O.
  3. Identification of Entamoeba histolytica / E. Dispar by the fresh amoeba technique vs its staining with hematoxylin-eosin in acute diarrhea. Rev Mex Pediatr 2005; 72 (3); 109-112. Available at: medigraphic.com
  4. Walwyn V, Iglesias M, Almarales M, Acosta N, Mera A, Cabrejas M. Usefulness of histological techniques for the diagnosis of infection in anatomical specimens. Rev Cub Med Mil , 2004; 33 (2). Available at: scielo.sld
  5. PanReac AppliChem ITW Reagents. Hematoxylin-eosin staining. 2017, Spain. Available at: itwreagents.com
  6. “Eosin.”  Wikipedia, The Free Encyclopedia . 7 Nov 2018, 08:18 UTC. 4 Aug 2019, 22:13 en.wikipedia.org
  7. “Hematoxylin.”  Wikipedia, The Free Encyclopedia . 3 May 2019, 11:23 UTC. 4 Aug 2019, 22:48 wikipedia.org

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