The Hematoxylin in the hematoxylin and eosin stain (H & E) is invariably an aluminum mordanted solution (a hemalum). It would be possible to use an iron-mordanted variation, but there would be little point to doing so as iron hematoxylin variants are often less stable or more difficult to use. The difference in color, blue-black compared to blue-purple, may be an advantage on occasion but other than that there would be no particular advantage.
Hematoxylin Types
Hemalums are usually classified as either progressive or regressive, the progressive requiring no differentiation and the regressive requiring differentiation to varying degrees. Rather than thinking of these as unrelated, they can be viewed as components of a continuum, beginning with the highly selective nuclear staining variants like Krutsay’s, and ending with variants like Ehrlich’s which stain the background quite darkly. In between the extremes are solutions that give high degrees of nuclear selectivity, with almost any degree of background staining that might be wanted. Given the large number of formulas available, it should be possible to choose one with the characteristics desired. If not, one could be designed based on the principles outlined in the discussion on formulating alum hematoxylin.
A common question is whether one type is better than the other for general staining. Although many technologists do have a preference, whether one is “better” or the “best” is extremely subjective. This discussion focuses on how to obtain a particular appearance rather than debating which appearance is preferable.
In general, progressive solutions tend to give slightly cleaner backgrounds with little or no mucin staining, although some of the higher hematoxylin variants (1.5 – 2 grams per liter) which do not contain any acid may do so to a fair degree. One of their best features is that relatively inexperienced technologists can more easily produce consistent results by adhering to a strict staining time, a desirable quality in some laboratories.
Regressive solutions, on the other hand, often stain mucin quite deeply, especially if they have a high concentration of hematoxylin (5 grams per liter or more) and are well-ripened. Some technologists also consider nuclear staining to be sharper following the differentiation of a regressive stain. Certainly, with these it is more convenient to alter the staining pattern in consecutive sections by differentiating for a shorter or longer time, leaving more dye for a darker background or less for greater contrast between nuclei and cytoplasm.
For many technologists, however, they are just two different ways to get the same result. There is no right way or wrong way in this. It is personal preference and convenience in getting the stain done satisfactorily. It is likely that more laboratories use a regressive procedure for routine staining, but this is often because it has usually been done that way and there is resistance to change, especially when it may result in a different appearance.
Acid Content
Hemalum solutions may, or may not, incorporate an acid in their formula. Ammonium and potassium alums, the two most commonly used, have a mildly acidic pH at a concentration of about 5% w/v, which is about the concentration in most formulae. Their buffering capacity, however, is quite limited and the pH will soon rise as the hemalum solution is used, especially if alkaline tap water is the preceding fluid. Rinsing sections in distilled water or dilute alum solution before placing them in the hemalum can reduce contamination and extend the life of the hemalum. If a non-acidified hemalum is the choice, this is strongly recommended.
Many laboratories use an acidified formula, so the contamination from the previous fluid is not so important. It should be noted, however, that a rinse in distilled water or alum solution would still be beneficial as it would extend the working life of a hemalum significantly. The useful life is often limited by alkaline carry-over of this kind rather than depletion of mordant or dye, and reducing carry-over can have a distinct effect. A fairly common practice, in fact, is to add a small amount of acid to a hemalum when it shows the first signs of exhaustion – a change in color from cherry red to red-purple. This rejuvenates the solution and often permits as many sections to be stained as were done initially.
Acetic acid is most commonly used in hemalums, at about 2%-5% v/v. A few formulae require other acids. Those of the Mayer type often use citric acid at 0.1% w/v, and this appears to be about equal in effect to 5% acetic acid. At least one formula uses 0.5% v/v hydrochloric acid. This formula has a very low pH and staining is essentially limited to DNA. That type of strong acid also removes calcium deposits, so they are not stained as they would be with acetic and citric acid-containing solutions.
Why choose an acidified hemalum rather than an unacidified one? It comes down to the type of staining wanted, and the convenience of obtaining it. Although the primary reason for designating a hemalum as progressive or regressive is based on the relative amounts of dye and mordant, a high ratio being more likely to be progressive, the presence of an acid can sharpen the staining characteristics, particularly with progressive formulae. An unacidified formula is more likely to stain the background distinctly, and the addition of acid can reduce this. With regressive formulae, the acid often has no visual effect but does affect how many sections can be stained before the hemalum needs to be replaced or rejuvenated.
Staining Time
Recommended staining times can vary from a minute or two to 30 minutes or so, depending on the formulation. There are some advantages to choosing a formulation that is not too fast acting. Very short application times run the risk of patchy and inconsistent staining. At least a few minutes are needed, preferably with some agitation to remove the reagents from the preceding steps. These can dilute out the hemalum locally and lessen staining in the vicinity, thus causing uneven coloration. Two to three minutes application with an initial 15 seconds of agitation will usually accomplish this. Without agitation, it would be preferable to extend staining to a minimum of 5 minutes, preferably longer, in a strong formula.
It is customary with progressive solutions to control the time so that consistent depth of staining is obtained. Commonly, for solutions such as Mayer’s or Cole’s, a staining time of 5-10 minutes is used. Progressive staining solutions are fairly tolerant of minor variations in the application time, and a few minutes will not usually make much of a difference once a significant degree of staining has taken place. Progressive solutions initially tend to stain rapidly to a point determined by the relative amounts of dye and mordant, then to self-limit further staining. This is not an absolute, as staining does intensify, but it does so at a much slower rate than the initial coloration.
Regressive application times are often applied within the range of 5-10 minutes as well, although some require longer times (Ehrlich, Delafield). The actual time is less critical than with progressive formulations since any excess can be removed easily with acid ethanol during the differentiation step. It is not uncommon to greatly overstain then extend differentiation to achieve the desired depth of color, and it is often convenient to do so. Patchy and uneven staining is rarely seen when used in this fashion unless there are difficulties with the sections.
When a regressive solution is used to stain progressively, as is sometimes recommended, then staining times are critical. A case in point is Harris’ hematoxylin. It is often stated that this solution can stain progressively if applied for a short time (about a minute). This may be overly optimistic but, when used this way, the timing is critical and a few seconds can make a significant difference in the depth of background staining. Due to the likelihood of inconsistent staining, short application times like this must be accompanied by significant agitation to ensure all of the tissue is bathed in full-strength hemalum as quickly as possible.
Differentiation
Although differentiation can be accomplished in several different ways, the usual method for hemalum is acid ethanol. This is usually 0.5% – 1% hydrochloric acid in 70% ethanol (standard acid alcohol), although for some purposes concentrations outside that range are used. Light differentiation on a staining machine where each step is applied for a fixed time of 30-45 seconds, for instance, can be accomplished with 0.05% hydrochloric acid. Whether a stronger or weaker acid alcohol is used makes little difference, within reason, except to the speed with which the dye is removed. If done correctly, both are capable of producing quality results.
Other acids may also be used. Although hydrochloric is the commonest, almost any acid which can dissolve in 70% ethanol could be used. Of course, different acids ionize to a greater or lesser degree and this can affect how efficiently they remove the dye. This should be determined by trials on test sections. The placenta is often a good test tissue for H & E stains. Very weak acids are often poor differentiators, whereas strong acids need to be diluted considerably. It should also be noted that there may be potential for interaction between the acid chosen and a component of the stain.
The subject of the solvent is often raised, as to why 70% ethanol is used instead of water. In fact, water can be used to dilute the acid, and will effectively differentiate the hemalum. It has been observed, however, that greater agitation is required to obtain the same degree of even dye removal. This is likely related to the currents generated when ethanol and water mix, an acid ethanol causing enough swirling around the chromatin to effectively wash away the initial floods of dye. The disadvantage of using water is that if there is no increase in agitation uneven staining is more likely. If agitation is increased then there is a greater possibility that sections may become detached from the slide. Notwithstanding that, water can be effective although ethanol is clearly superior. Although other alcohols such as n- or iso-propanol are sometimes used quite effectively as a substitute, ethanol remains the commonest.
Blueing
When applied from an acid medium, as most hemalums are, the initial color of the material stained is brick red. This is not permanent and must be changed to blue. The red phase of alum-hematein is soluble, and will slowly leach from the structures into whatever medium is used under the coverslip. The blue phase is insoluble and is generally considered to be permanent. The change is made by altering the pH. It is customary to place the stained and differentiated sections into an alkaline medium for a few minutes to convert the stain, then to wash with water to remove whatever was used to alter the pH.
In many parts of the world, the water is alkaline, and this may be perfectly satisfactory for blueing. In other places, the water is acidic and some other means of conversion is required. The simplest means is to add a few drops of strong ammonia to some water until it has a pH of about 9-10, then place the sections in it until they are evenly blue. Other suitable blueing agents are 0.1-1% lithium carbonate solution, 0.5% sodium acetate, 2% sodium bicarbonate, or a more complex solution such as Scott’s tap water substitute.
Some municipalities also add chemicals to their water supply. Usually, this is a chlorine compound and is added to ensure the water is pathogen free. At some times of the year, the amount may be increased and this can have an effect on the hemalum staining. Chlorine compounds are notorious bleaches and residual traces of chlorine may lead to fading. If this is the case, then excessive washing should be avoided and a rinse with chlorine-free water before proceeding would be of benefit.
Following blueing in a dilute alkaline solution the sections must be washed to remove the excess blueing agent. When this is done it is important to ensure that the wash water itself is not acidic and does not reconvert the blue hemalum back to red, even partially. Distilled water is usually satisfactory but, if not, a very small amount of an alkalizing agent may need to be added. The pH should be just on the alkaline side of neutrality, but if the wash water is too alkaline then subsequent staining with eosin can be inhibited.
When large amounts of such water are required a container of suitable volume with a layer of marble chips or crushed oyster shells on the bottom can be filled with tap water, perhaps controlled with a ballcock or similar automatic filling system. If the container is elevated the water may be drawn off by gravity through an opening in the base, ensuring that it flows over the calcium carbonate. Slightly alkaline water will then always be available in volume.
