Web Based Promotion! 10% Off any initial product order. Mention promo code 1204
Volume 5, Number 1
Spring 2002

Searching for Helicobacter
Figure 1. Gastric biopsy fixed in Prefer, then stained with Hp Yellow and Hp Blue, our new procedure to replace Alcian yellow and toluidine blue. Helicobacter pylori stain conspicuously against a yellow background. 100x

Welcome...

The last issue of The Innovator addressed the disappearance of Alcian blue, problems of availability and quality with nuclear fast red, and the desire for a non-carcinogenic alternative to Congo red. While we developed new products to meet those needs, we also started work on another project involving Alcian yellow, an important dye used in detecting Helicobacter. The world supply of Alcian yellow was gone, for much the same reasons that marked the demise of Alcian blue late in 2000. Unlike Alcian blue, we were unable to bring Alcian yellow back.

This is the story of how we found a way around the problem. As so often is the case, the new process has significant advantages over the original while delivering comparable results.

Helicobacter pyloriis the probable causative agent of gastric ulcers and as such is commonly sought in gastric biopsies. These bacteria are hard to see with most stain because they are embedded in mucus and mucous cap cells which line the stomach.Apparently negative specimens may in fact contain bacteria hidden by a low-contrast stain. A new procedure is now available that takes the guesswork out of the search. It is fast, economical and environmentally acceptable. More to the point, it is available. See for yourselfhow easy it is to find H. pylori (Figure 1)!

If you think the specimen in Figure 1 looks like it was stained with Alcian yellow and Toluidine blue (AY+TB), you are close (and we are happy). The disappearance of Alcian yellow from commercial markets prompted us to develop an alternative procedure to mimic this important stain. While silver stains demonstrate H. pylori well, they are capricious, time consuming, expensive and environmentally hazardous. Immunohistochemistry is invaluable for difficult cases, but is much too expensive and time consuming to be used as an initial screening device. The AY+TB method of Leung et al (1996) has proven to be the premier way to stain these bacteria (Rhatigan-Drexler, 1999); regrettably it is no longer possible without Alcian yellow.
Table 1 compares the four steps in each protocol. Remember that mucus in and near the surface of the stomach lining is neutral and will not stain directly with basic dyes. In both techniques, periodic acid oxidizes the hydroxyls in carbohydrates (mucus) to aldehydes. Sodium metabisulfite then attacks the aldehydes to create sulfonic acids in the AY+TB stain. Mucus is now highly acidic and stains readily and nearly irreversibly with Alcian yellow. Toluidine blue at high pH then stains bacteria and most tissue elements except yellow mucus. Because bacteria are restricted to yellow mucus within and upon the surface of mucous cap cells, they stand out clearly. Alcian yellow has blocked the acidic sites in the mucus, and cannot be displaced by toluidine blue. Further, Alcian yellow is insoluble in higher alcohol, so it is not removed during dehydration; toluidine blue (and nearly all other basic dyes) are extracted in higher alcohol solutions.

We tried synthesizing Alcian yellow using the same proprietary technology behind our Alcian blue (Anatech Ltd., 2001), but we could not obtain raw materials. Compounding the problem, no other yellow dye has the same bonding properties as Alcian yellow. Other dyes stained mucus yellow but could not resist displacement by toluidine blue or loss during dehydration. We finally turned to the reactions behind the AY+TB procedure to see if alternative chemical pathways would produce similar results. Two new stains, Hp Yellow and Hp Blue are the products from that research: together, these Hp stains (Hp for H. pylori) duplicate the colors from AY+TB.

Figure 2.
Both toluidine blue (left) and Hp Yellow (right) have similar flat intermeshed ring systems that allow the molecules to stack together, held by van der Waal's forces. Yellow mucus then stains green.

In the Hp procedure, a Schiff-type reaction is used to stain aldehydes created by periodic acid. Hp Yellow is a Schiff reagent of sorts: it is highly specific for aldehydes, but is not colorless like conventional Schiff made from basic fuchsin. It also bears a positive charge and acts like a basic dye, staining everything in the specimen that is negatively charged. Step 3, acetic acid, simply destains all ionically bound Hp Yellow, leaving covalently bound mucus yellow. Thus, we achieve the same result using an entirely different chemical reaction. Bacteria, nuclei and cytoplasm are then stained with Hp Blue.
Figure 3.
Gastric biopsy fixed in NBF then stained with Hp Yellow and Toluidine blue. Mucus tends to have a greenish cast but bacteria are still visible at the tip.
Why not use toluidine blue? We tried, and it does work although mucus stains greenish yellow or green in some specimens. Because Hp Yellow and toluidine blue are so similar in molecular shape (Figure 2), they stick together via dispersion forces (a type of van der Waal's attraction) and cannot be dislodged. The degree of greenness varies from specimen to specimen, apparently as a result of different fixatives, fixation times and other processing variables. Neutral buffered formalin tends to produce the greenest hues, Prefer the best yellow. Careful selection of staining times for the two dyes can minimize the green effect. Even when mucus is definitely green, bacteria still stand out (Figure 3). However, Hp Blue is a better choice, especially if tissues are fixed in formalin. Incidentally, toluidine blue does not bond to Alcian yellow because their shapes are sufficiently different to preclude stacking.
Figure 4.Hp Blue (left) is very different from Hp Yellow (right): rings are widely separated by an azo bridge (paired blue atoms).

Figure 5.

For those who prefer paler colors than seen in Figure 1, simply reduce staining time in Hp Yellow. Either way, the bacteria stand out clearly in the free mucus and within the glands. 40x

Because nearly all blue basic dyes are shaped just like toluidine blue, we had to reach far beyond the biological field to find one that was sufficiently distinct that it could not bond to Hp Yellow. Since it has no common name, we call it Hp Blue (CI 11105). Its shape is very different from Hp Yellow (as dyes go, anyway; see Figure 4).

Staining times for the Hp procedure are similar to those in the AY+TB sequence: 10 minutes in periodic acid, 45-120 seconds in Hp Yellow, 2 minutes in acetic acid and 2-4 minutes in Hp Blue. Varying the times within these ranges allows you to obtain delicate to bold results, compare Figure 5 (below) and Figure 1 (top of page). Bacteria are obvious, even at low power. (Figure 6, below ).

The original AY + TB stain had two shortcomings aside from the problem of availability. Alcian yllow was poorly soluble at the prescribed concentration and most of this precious dye was simply lost on the filter paper. Toluidine blue is not stable at high pH (10) and cannot be made in advanceof staining by more than a few hours. Hp Yellow and Hp Blue are stable solutions, ready to use. Complete directions for staining nd troubleshooting are available with each bottle of stain.

References

Anatech Ltd., 2001. Three dyes, three dilemmas.
The Innovator 4(2):1-4
Leung JK, KJ Gibbon and RK Vartanian, 1996. Rapid staining method for Helicobacter Pylori in gastric biopsies.
J Histotechnol 19:131-132.
Rhatigan-Drexler K, 1999. A comparison of staining methods for Helicobacter pylori. HistoLogic 30:3-8.
Figure 6.

Cursory scanning at low power may reveal the bacteria in high contrast to their yellow background, as it does here. for a closer look, see Figure 7. Gastric biopsy fixed in Prefer and stained with Hp stains. 20 x

Figure 7. Need we say more? Gastric biopsy fixed in Prefer and stained with the Hp stains. 100x