Haematoxylin Staining Solutions and Methods

Haematoxylin Staining Solutions and Methods

Haematoxylin Staining Solutions and Methods

Haematoxylin solutions can be classified acyl cording to the type of mordant used. The following are the common haematoxylin stains based on the mordants used.

1. Alum haematoxylin

2. Iron haematoxylin

3. Tungsten haematoxylin

4. Lead haematoxylin

5. Molybdenum haematoxylin

6. Haematoxylin without mordant

Alum haematoxylins These are the most routinely used haematoxylins. They produce good nuclear staining. The mordant is generally aluminium in the form of ammonium alum or potash alum. The nuclei are stained red which are converted to blue-black, by washing in a weak alkali solution or tap water (blueing). Scott's tap water is frequently used for blueing. The most widely utilised alum haematoxylins are the Ehrlich's, Mayer's, Cole's and Harris's.

 Iron haematoxylins The most commonly used iron salts are the ferric chloride and ferric ammonium sulphate (iron alum). These iron salts serve as both oxidising agents and mordants. The ferric salts oxidise the haematoxylin chemically and so should not be kept for too long. Ideally, it should be prepared immediately before use.

Iron haematoxylins demonstrate a wider range of tissue structures than the alum haematoxylins but are more time-consuming. Examples of iron haematoxylin are the Weigert's, Verhoeff, Loyez and Heidenhain haematoxylins. 

Tungsten haematoxylin The phosphotungstic acid haematoxylin method of Mallory is the only application of tungsten haematoxylin, others being a variation of this method. It is employed to demonstrate many tissue structures, but it is particularly useful for fibrin, muscle striations, cilia and glial fibres. 

Lead haematoxylin Lead nitrate is the mordant for this haematoxylin. It is commonly used to demonstrate the granules of the endocrine cells of the alimentary tract. It is particularly useful in the study of localisation of gastrin secreting cells in the stomach. 

Molybdenum haematoxylin This rarely used haematoxylin uses molybdic acid as a mordant. Its limited value is in the demonstration of argentaffin cell granules which are usually shown by other methods. 

Haematoxylin without mordant This group of haematoxylins is no longer in common use. They were useful in the demonstration of various minerals such as lead, iron and copper in tissues. The method was introduced by Mallory.

PREPARATION OF HAEMATOXYLIN STAIN SOLUTIONS

Iron Haematoxylin

(a) Weigert's iron haematoxylin This stain is widely used in conjunction with Van Gieson's stain for differentiating muscle fibres and connective tissue.

 Solution A

Haematoxylin	1g
Ethyl alcohol, 95%	100ml

Solution B

Ferric chloride, 29% aqueous solution	4 ml
Hydrochloric acid	1 ml
Distilled water	95 ml

Working solution 

Solution A 1 Vol 

Solution B 1 Vol

 Mix the solutions well to give a deep purple colour and use within 48 hours. 

 (b) Heidenhain's iron haematoxylin This is a very useful stain for demonstrating both nuclear and cytoplasmic inclusions. It is also used to demonstrate muscle striations. 

Solution A (Mordant) 

Iron alum	2.5 g
Distilled water	100 ml

Solution B

Haematoxylin	0.5g
95% ethyl alcohol	10 ml
Distilled water	90 ml

The haematoxylin is dissolved in the alcohol and then added to the water. The stain is left for 4-5 weeks to ripen. The stain is stable for a long time.

Alum Haematoxylins

There are many haematoxylin stains which use alum as mordant. 

(a) Mayer's acid alum haematoxylin This is a general-purpose stain

Ammonium alum	50g
Chloral hydrate	50g
Haematoxylin	1g
Citric acid	1g
Sodium iodate	0.2 g
Distilled water	1000 ml

With the aid of gentle heat, the haematoxylin is dissolved in water. The sodium iodate is added, followed by the alum. The citric acid and the chloral hydrate are then dissolved. The stain is ready for immediate use as no ripening is required. This stain is stable for several months. 

(b) Ehrlich's haematoxylin This is a very useful all-purpose nuclear stain

Ammonium or potassium alum	3 g
Haematoxylin	2 g
Ethyl alcohol 95%	100 ml
Glycerol	100 ml
Distilled water	100 ml
Glacial acetic acid	10 ml

The haematoxylin is dissolved in the alcohol before the addition of the other ingredients. The stain may be ripened naturally when allowed to stand in a large container, loosely stoppered with cotton wool, at room temperature and exposed to direct sunlight. The flask should be shaken frequently, and ripening takes a few weeks. When a test slide gives a good staining effect, the stain is bottled and filtered before use. It is also possible to ripen or oxidise the stain and use it immediately by adding 0.3 g sodium iodate to the stain.

(c) Harris haematoxylin This is a powerful selective nuclear stain. It is widely used in exfoliative cytology as a nuclear stain because it gives sharp delineation of nuclear structures. Staining time is usually 2-5 minutes. 

Haematoxylin	1g
Absolute alcohol	10 ml
Ammonium or potassium alum	20 g
Distilled water	200 ml
Mercuric oxide	0.5 g

Dissolve the alum in hot water, dissolve the haematoxylin in absolute alcohol, and add it to the alum solution. Bring quickly to a boil and add the mercuric oxide which turns the solution dark purple. Cool rapidly under the tap. The addition of about 8.0 ml glacial acetic acid to the stain is recommended to sharpen nuclear staining. This stain, which is normally used regressively, should be prepared in a flask of ample size due to the frothing that occurs when the mercuric oxide is added. It should be filtered before use. 

(d) Cole's hematoxylin though not very popular, Cole's haematoxylin gives satisfactory general staining effect. Unlike Ehrlich's haematoxylin, it is suitable for use in sequence with celestine blue. 

Haematoxylin	1.5 g
1% iodine in 95% alcohol	50 ml
Saturated aqueous ammonium alum	700 ml
Distilled water	250 ml

Dissolve the haematoxylin warm distilled water, mix with the iodine solution. Add the alum solution and bring to a boil. Cool rapidly and filter before use.

Eosins

Eosins are acid xanthene or phthalein dyes. Eosin Y eosin B, phloxine and erythrosin (which unlike other eosins, is halogenated with iodine), are the common members of this group of dyes. Eosin is derived from fluorescein and is available in two main shades--yellowish or blueish. When properly used on a well-fixed section it stains connective tissues and cytoplasm in varying colour intensity and shades of the primary colour. It is most commonly employed as a contrast stain because it gives a useful differential contrast to nuclear stains. With haematoxylin, eosin is the routine counterstain in histopathology.

Eosin Y (yellowish) is the most frequently used and is readily soluble (44% w/v in water and 2% w/v in ethanol). The stock aqueous solution is generally made up in 5% w/w concentration, using tap water or distilled water. The alkalinity of tap water is considered to give better staining effect. A crystal of thymol or 0.25 ml of formalin is added to each 100 ml of stock to prevent the growth of moulds. Moulds may also be removed by filtration and they do not affect the stain. The aqueous stain is usually used at 1% for 30 seconds to 5 minutes, depending on the type of fixative, tissue and intensity of colour desired. The exact strength of the working solution is not critical.

The 1% w/v alcoholic solution is prepared by dissolving lg eosin in 20 ml distilled water and made

up to 100 ml by adding 80 ml of ethanol. The working solution maybe 0.25% or 0.5% diluted with 80% ethanol. Some workers claim that the addition of 0.2 ml glacial acetic acid to the aqueous solution or 0.5 ml glacial acetic acid to the alcoholic solution enhances the intensity and selectivity of eosin.

Eosin is the most commonly used counterstain in haematoxylin staining methods. However, many substitutes are available, e.g., phloxine, Biebrich Scarlet, erythrosin and orange G. These substitutes are prepared in similar concentrations and modes as eosin.

Routine Haematoxylin and Eosin (H&E) Staining Method

The haematoxylin and eosin (H&E staining technique is performed to demo iterate the general structure of tissues. It is the recommended routine stain to be done on all tissues.

Procedure

1. Take section down to the water (Refer to the theory of staining technique).

2. Stain in haematoxylin solution for 5-10 minutes.

3. Rinse in water for a few seconds.

4. Differentiate in 1% acid alcohol with continuous agitation for 10-15 seconds.

5. Wash in running tap water (blueing) for 5 minutes.

6. Stain in 1% aqueous eosin solution for 5 minutes.

7. Wash in running tap water for 30 seconds.

8. Dehydrate, clear and mount.

Result

Nuclei - bright blue

Cytoplasm, collagen - pale pink

Muscle, keratin and colloid - bright pink

Erythrocytes - orange-red

Note

1. To stain many slides, arrange them in a suitable bottomless glass slide carrier, one slide in each slot.

2. Alcoholic eosin solution can also be used.