Hematoxylin Staining
Some Technical Notes

Paul D. Millikin, MD

The word hematoxylin is derived from the old Greek words Haimato (blood) and Xylon (wood), referring to its dark red color in the natural state, and to its method of manufacture from wood.

Hematoxylin (C.I. 75290) is a natural dye extracted by boiling the wood of the South American and West Indian logwood tree (Haematoxylon campechianum), and partly purified by recrystallization. It is sold commercially as a crude mixture of hematoxylin and other, unidentified substances. It comes as a brownish tan powder which is poorly soluble in water and somewhat more soluble in ethyl alcohol (1 gm/100ml water and 30-40 gm/100 ml alcohol). The active dye is not hematoxylin itself, but its oxidized product, hematein. The latter can be bought, but it is expensive, and the same results can be had simply by oxidizing the crude hematoxylin.

In the early days, oxidation was carried out by making up the hematoxylin solution, plugging the container with cotton, and leaving it exposed to light and air for 6 weeks to several months.

However, it was soon found that oxidation (also called ripening) could be achieved much faster by adding a wide variety of oxidizing agents, the two most common being sodium iodate and mercuric oxide. With either agent, oxidation not only begins immediately, but apparently continues for some time, causing dye precipitates of uncertain composition, and eventually exhausting the stain.

The addition of glycerin to several formulas is said to guard against over-oxidation and perhaps to retard fungal growth.

 

Hematoxylin lakes
Used alone, hematoxylin is a poor stain, but in combination with various metallic salts (mordants) which couple it to the tissue, it is one of the best nuclear stains known. It has also been used to detect metals in tissues, to stain myelin, to stain mitotic figures, fibrin, muscle cross striations and other tissue elements.

The combination of hematoxylin plus mordant is called a hematoxylin lake, and lakes with different metals have different colors. The aluminum lake formed with ammonium alum (aluminum ammonium sulfate) is particularly useful for staining nuclei. It is purple in acid solution, but blue in alkaline solution. Instead of aluminum ammonium sulphate, either aluminum potassium sulphate (potassium alum) or aluminum sodium sulphate (sodium alum) may be used to create the aluminum lake. No appreciable difference in results is found. Hematoxylin recipes using any of these mordants are loosely called alum hematoxylins, or hemalums.

Other hematoxylin lakes are used for different staining purposes, and have a variety of colors, depending on the metal used.

 

Numerous formulas for alum hematoxylin have been devised ever since Boehmer first described it in 1865. Some of the more common are listed elsewhere in StainsFile.

The staining of nuclei by alum hematoxylin is enhanced by the addition of acetic acid, which apparently reacts with the nuclear chromatin, giving it a somewhat crisper appearance. However, because of the alum content, the pH of alum hematoxylins tends to be around 2.6–2.9 even before the acid is added.

 

Progressive vs regressive staining
All alum hematoxylins, whatever their formula may be used as either progressive or regressive stains.

Progressive staining means that the tissue is left in the stain just long enough to reach the proper endpoint. Therefore it may be necessary to examine the slides at several different intervals to determine when staining is dark enough but not too dark.

Regressive staining means that the tissue is deliberately overstained and then destained (differentiated) until the proper endpoint is reached.

The difference between methods is largely one of convenience. Progressive hematoxylins are generally less concentrated and work slowly to avoid overshooting the endpoint.

Regressive hematoxylins are more concentrated and many can achieve overstaining in a matter of less than a minute, while differentiation (removal of excess stain) requires only a few seconds. Also, timing is not so important in regressive procedures. As long as the slide is overstained, it doesn't matter whether it was in the staining dish for 1 minute or 10.

Regressive procedures are therefore faster and more convenient than the progressive ones, and they have the added advantage that differentiation also removes hematoxylin from the gelatin or other slide adhesive, producing a clear, transparent background.

Differentiation is done in dilute acid (usually acid alcohol because of hematoxylin's greater solubility in alcohol). Differentiation is stopped immediately by simply washing the slides in water.

If too much hematoxylin has been removed, a few more seconds in the stain will correct the problem, and the whole process can be repeated.

If too little has been removed, a few more dips in acid alcohol will produce the correct endpoint.

Note: With all hematoxylins, progressive or regressive, the endpoint is the same!

In a properly stained slide, only the nuclei, flecks of calcium, and certain kinds of mucus will be stained, while the background will be unstained or very pale brown. There should be no hint of blue or gray.

 

Blueing
Because most alum hematoxylin formulae are fairly acid, the nuclei will at first be stained the purplish color of the acid dye. Changing their color to blue gives a much better contrast with the usual red counterstains.

When the endpoint has been reached by either progressive or regressive methods, nuclear color can be changed in one of two ways.

  1. The slides may be dipped for a few seconds into a weakly alkaline solution such as ammonia water or dilute sodium carbonate. Note that differentiation stopped the second you rinsed the slide in water, so the alkaline solution is not necessary for that.
  2. They may be washed for 2-5 minutes in tap water. Depending on the geographic area and the local method of water treatment, tap water tends to be slightly acid, with a pH in the range of 6.0 - 6.8. However, this is considerably more alkaline than the pH of most alum hematoxylins (2.6 - 2.9), so bluing results. The tap water wash has the added advantage that it washes out any excess alum, giving a crisper nuclear stain and preventing fading during storage.

 

Variability of hematoxylin

  1. The strength of all hematoxylins varies from day to day, becoming a little weaker with every use because the slide racks carry a little water over with them, causing some degree of dilution. Also the strength becomes stronger each time a new solution is made.
  2. Oxidation continues slowly and irregularly from day to day, further reducing the dye strength by producing unpredictable amounts of precipitated dye which must be filtered out.
  3. Oxidation produces several oxidized derivatives of hematein, from monoxyhematein to pentoxyhematein, each with a different color. The di- and tri-oxy derivatives appear to offer the optimum color. Tetroxyhematein is brownish and pentoxyhematein is colorless, so obviously the oxidizing step can be carried too far and render the dye unusable.
  4. To slow down aerobic oxidation of the stock solution in storage, some people cover the surface with a layer of oil and pipette from below the surface when more stain is needed.
  5. Remember that the original hematoxylin was a crude extract of logwood, so it may contain hundreds of unknown substances besides hematoxylin, and some of them and their oxidized products may be responsible for some of the precipitates.
  6. Note the "dye content" on the label of the jar containing the hematoxylin powder. A dye content of 50% means that only half of the powder is hematoxylin, and the other half is a mixture of unknown composition.
  7. The precipitates may cause obstruction in some mechanical stainers if the dye must pass through tubes or orifices of small caliber.

 

Eosin staining
The principles just outlined for hematoxylin staining also apply to eosin Y or the other red counterstains, except that the endpoint is not quite as sharp. A good counterstain will not only contrast sharply with the blue nuclei, but it will allow the non-nuclear tissue components to be clearly differentiated from each other; smooth muscle from collagen, for example.

Note that eosin Y is normally dissolved in 95% ethanol. Therefore an eosin-stained slide will be decolorized if it is left very long in 95% ethanol before coverslipping. Eosin solubility in 100% ethanol is considerably less, but here, too, stain can be lost slowly.

To avoid these problems, slides should be run quickly from the eosin dish to the clearant dish. If you use a staining rack, you will find that 7-10 "sloshes" in 95% ethanol will probably be sufficient, before advancing into 100% ethanol, and then quickly into clearant to avoid any eosin loss.

The shade of the red counterstain is important for optimum contrast with the blue nuclei. Eosin Y normally has a somewhat yellow-orange color, but adding a little acetic acid to the solution will cause it to become redder. That improves the contrast with the blue nuclei. Be careful not to add too much acid, however, as it can reduce or even eliminate the contrast due to intensity differences between tissues stained with eosin Y.

The density of the red color is important for differentiating the non-nuclear components. Too little color will make the slides look pale and washed out and will cause a certain amount of glare. On the other hand, too much dye will blur the distinction between the non-nuclear tissue elements.

 

Judging endpoints

  1. The endpoints of both hematoxylin and eosin staining involve color density and contrast, neither of which is measurable by a clock.
  2. The human eye, however, is very good at both measurements.
  3. Therefore an experienced tech will throw away the clock, no matter what the textbook says, and use the eye instead.
  4. At first, endpoint judgment will have to be under the microscope, which will take a little time.
  5. However, with a little experience and a standardized control slide, judgment can be made accurately by the naked eye in just a few seconds.

 

Final note
Many professional histotechnologists who won't settle for less than the best, even with their routine H&E slides, add a control section of normal appendix to every slide rack every day. There are plenty of nuclei in the lymphocytes and plasma cells for judging the hematoxylin endpoint, while the submucosal collagen and adjacent smooth muscle serve very well for judging the eosin endpoint.

When the hematoxylin endpoint has been reached on the control slide, the whole rack is considered properly stained.

If the endpoint is incorrect, the whole rack of slides is restained until the appendiceal nuclei are properly colored.

Only after the proper hematoxylin endpoint has been reached do they proceed to the Eosin dish, where the control procedure is repeated. After the whole rack of eosin stained slides has been run up to clearant, the endpoint of eosin staining is judged by the control appendix.

If it is not correct, the whole rack is restained. After the eosin endpoint is correct, the whole rack of slides is ready for coverslips, and not before.

Does that sound like too much trouble? Remember that any histotech who has learned to judge endpoints through the microscope rapidly learns to judge them with the naked eye.

When that level of experience has been reached, a standard rack of 20 slides can be stained properly, and beautifully, in less than 10 minutes.

Admittedly, not all tissues stain exactly like appendices, so any problem slides should be stained individually, using the microscope!

If you aren't willing to settle for just adequate, but want your slides to be uniformly gorgeous, try using your eyes instead of your clock.

 


 

Translate in
Google Translate
Instructions