H & E – The Eosin
The second component of the H & E is eosin, the counterstain. There are several dyes named eosin, but the one most commonly used to counterstain hemalum is eosin Y. This dye is both water and ethanol soluble. Eosin Y is a pink, acid dye which also displays yellow-green fluorescence. This can be seen with white light, although it is stronger with ultra-violet, and it has led to some difficulties with colour film recording in years past. A related dye sometimes used is ethyl eosin. This is alcohol soluble. Another choice is eosin B, this is also water and ethanol soluble, but has a bluish cast to it. It is not a popular choice for sections, although it is useful in Romanowsky stains.
There are also a few other dyes that have been used, often due to the difficulties that older colour film had in recording the colour of eosin Y. Modern colour film and digital recording techniques do not have this difficulty, so their use has declined. Phloxine B, erythrosin B and rose bengal are the ones closest in appearance to eosin. Mercurochrome was used to mark tissue margins, and rarely as a counterstain. However, it should no longer be used for either due to its mercury content. Tattoo inks are better for margins, with a wider colour choice. See a comparison of these dyes.
Eosin, like many dyes, can be used both progressively or regressively. Many technologists believe that the most differential appearance is obtained by overstaining (about two minutes) with a solution of about 2% concentration, then washing to remove excess. When this is done effectively, then four different intensities of colour can be seen. The most intense is eosinophils which will be almost iridescent, followed by bright pink erythrocytes. Muscle will be paler than erythrocytes, but still distinctly pink, and collagen will be quite pale coloured. It does take some practice to get this effect, but it also requires the correct type of local water. This should be hard, with calcium salts. The most studied in this regard is probably tap water from London, England, where this way of eosin staining used to be standard. If your local water is similar to London's, then it may be the most effective way to apply eosin.
Other technologists prefer to stain progressively with an aqueous eosin solution. These should be more dilute than for regressive staining, often in the range of 0.1%-0.5%, and they are applied for around 30 seconds, followed by a brief rinse with water and dehydration. These times and the concentration of the dye should be established by trials, but once established and giving satisfactory results, will remain fairly consistent.
The fact is that the qualities of water available locally varies considerably throughout the world and ranges from strongly mineralised alkaline water to forest runoff with a distinct humic acid content. For that reason suitable tap water may not be available. If staining from an aqueous solution is wanted and acidic water is supplied locally, then a water treatment system mentioned in the page on hemalum staining may be suitable. That is a large container with a layer of marble chips or crushed oyster shells can be filled with tap water, perhaps controlled with a ballcock or similar automatic flow system, and slightly alkaline water will then always be available in volume. Otherwise, eosin may be dissolved in 2% calcium chloride solution in distilled water.
Many technologists working with unsuitable water supplies find a better resolution of staining difficulties is to use an ethanolic solution with either eosin Y or ethyl eosin. It should be noted that eosin Y, although often described as “water soluble eosin” is also ethanol soluble sufficiently to stain quite satisfactorily.
There are many variations on this model, but the procedure is no different in principle than staining from water. Progressively, a 0.1% eosin Y solution in 95% ethanol for 10-15 seconds is often satisfactory if followed by a brief rinse in 95% and absolute ethanols to remove excess. Applying for a shorter or a longer time will control the depth of staining for all practical purposes.
Alternatively, application may be regressive with the same or a stronger solution being applied for a minute or two then differentiated by washing for a period of time in 95% ethanol, followed by rinsing with absolute ethanol. The specific times should be established with trials carried out locally to determine the most suitable procedure. Once again, placental tissue is a suitable test material.
Although most reference texts do not recommend it, small amounts of acetic acid may be added to eosin solutions, both aqueous and ethanolic. Usually this is in the range of drops per litre and would likely be at a concentration of 0.01% or less. The acetic acid is an accentuator and intensifies staining, as acetic acid usually does with acid dyes. The purpose is to enhance staining intensity a little without completely overshadowing the nuclear stain nor turning it red. The amount added will determine how much the intensity is increased, but it will also decrease variations in tone and the differential effect of eosin may be lost. Once again, however, what constitutes a suitable depth of staining is quite subjective and trials should be run with different concentrations of both dye and acetic acid.
The three dyes, eosin Y, eosin B and ethyl eosin, are all very similar in staining characteristics but rose bengal, phloxine B, erythrosin B and mercurochrome are redder, and structures stained with them tend to stand out quite prominently. This has led to the practice of adding a small amount of one of these, usually phloxine B, to a solution of eosin Y. This is preferentially picked up by some structures, such as erythrocytes, and gives a pale pink overall colouration with distinctly more prominent staining in some others. Thus it mimics the effect obtained with eosin Y followed by water washing.
This practice arises from the use of acetic acid to intensify eosin staining. As it does this it also reduces the differential effect obtained with very mildly alkaline solutions. Adding a second and redder dye partially restores that effect or, at least, simulates it. Like most things, it can be overdone and not very much is needed to get the effect. A final concentration of 0.1% with eosin Y at 2% would be quite enough. The higher the concentration, the more the secondary dye will replace the eosin Y. Since the object is to obtain contrast, using too high a concentration would be counterproductive. Once again, trials with differing concentrations of the redder dye should be carried out to determine the optimal amount for the effect wanted.