H. pylori isn't as blue as it is painted

Giemsa staining for the determination of Helicobacter pylori is one of the most important staining in gastroenterology diagnostics. It helps in assessing the cause of changes in the stomach or duodenum. While the staining itself is not problematic for an experienced pathologist, due to its monochromatic nature, it can lead to headache by young diagnosticians. More and more often, immunohistochemical determinations are used, which, thanks to DAB+, mark bacteria in brown. However, compared to the histochemical methods, it is incomparably more expensive.

Today, after a very long time, I decided to write about the methods of this dying, because recently during intensive training provided in various laboratories I noticed that seemingly very simple dyeing can be quite problematic. Unfortunately, not everyone knows that the monochromatic staining of Giemsa usually results from strictly adhering to the guidelines described by suppliers who do not deal with techniques in pathology at all or their knowledge is negligible. Not only can we make a multi-color image of a stomach or duodenal biopsy from a simple staining, we also have a few staining that can significantly improve the assessment of H. pylori.

I will start today with a method that is not very popular, mainly due to Alcian yellow, which for a long time was very expensive and hardly available, but today this dyeing is much cheaper and more convenient (cat. 010269, DiaPath). Just check the picture below:

The method was proposed by J. K. Leung and K. Gibbon's in 1998 and to this day is the method that improves the evaluation of H. pylori the most. Many modifications of the Giemsa staining method produced by various manufacturers are based on the Leung & Gibbon's method.

To perform the original staining, we need 4 stock solutions:

• periodic acid 1%

• sodium disulfate 5%

• alcian yellow 1%

• toluidine blue 1%

Methodology:

1. The sections must first be deparafinised and hydrated

2. Oxidize in 1% periodic acid for 10 min.

3. Rinse well with tap water

4. Reduce to 5% sodium bisulfate for 5 min

5. Rinse with tap water

6. Dye with Alcian yellow for 5 min

7. Rinse off excess dye well in tap water

8. Stain with freshly prepared toluidine blue for 3 min.

9. Rinse excess dye in water.

10. Dry it

11. Dehydrate, clear and cover quickly.

For dehydration, we can use a xylene / acetone mixture in increasing ratio for xylene:

(xylene) 1: 3 (acetone)

(xylene) 1: 1 (acetone)

(xylene) 3: 1 (acetone)

Freshly prepared toluidine blue will provide us a pure solution with no additional bacteria that could contribute to false positives. Some of the reagents offered by the producers (cat. C0142, DiaPath) contain substances that prevent the survival and grow of fungi and bacteria, so they can also be used without preparing the reagents on an ongoing process.

The second staining worth mentioning as a curiosity, although practically unused, is the staining according to Dr. Sadie Sayeed's method. This method uses Schiff's reagent to stain the mucopolisaccharides with a magenta color. Nevertheless, the Schiff's reagent is very sensitive to changes in temperature, type of water used, etc. and can very quickly lose its staining properties, changing the color of the preparation from raspberry to purple, which in the case of blue color after staining with methylene blue results in a rather unattractive differentiation of structures.

Another staining that we can use in the evaluation of H. pylori is the GENTA staining (cat. 1660, Morphisto GmbH). Stain completely different from all available on the market, mainly because the background is orange/red and the bacteria are gray and black.

For dyeing we use, among others, mastic gum, silver nitrate in various concentrations, Gill hematoxylin III, alciane blue or hydroquinone. Before we start staining, we need to prepare base solutions and then we can get down to the essence of the matter. The entire part of the staining should be done at a temperature of 45-60 degrees C and the rest at room temperature. We also cannot just put slides into Coplin or Hallendahl jars, we must first heat them to a temperature of 45 degrees Celsius. With good news, as long as the original dyeing lasts 1.5 hours, the modification takes as little as 0.5 hour (excluding preparation of course).

Staining, from the diagnostic point of view, is very effective and simplifies diagnostics, but, from the technical point of view, it is very demanding. If you are interested I invite you to read the publication about this staining, the title of which is at the end.

The icing on the cake today is the standard Giemsa staining. The most common color of colored slides that we meet is blue. Possibly dark blue. However, by selecting the appropriate rinsing times in the dehydration phase, we can give the tissue a very interesting color. After all, Giemsa's dye is not a single-component reagent. It's dissolved methylene blue and eosin in alcohol. If we realize this, we immediately know that the colors we can get ranges from pink, through purple to navy blue. By selecting the appropriate length of rinsing in 70% alcohol after dyeing, we can achieve full color differentiation.

Why am I not giving any specific time? Because it does not depend only on time, but on who is the producer of Giemsa solution (exactly what is the concentration of eosin), how much minerals are in water you have, what concentration of ethyl alcohol you use. One thing is certain, keeping the preparations in ethyl alcohol for too long will make the color uniform, which will give us a navy blue effect.

Below You will find some well stained picture of Giemsa stain.

Next time You will make the Giemsa staining, please keep this picture in mind to reach this level. If You have it In Your lab then big congrats!

Other stainings for H. Pylori not mentioned in obove text, but worth to check:

• Warthin-Starry stain (faster method with silver nitrate, very popular)

• Field's stain (for Malaria, but with very thin sections 2-3 micrometers, also suitable for H. pylori

• Toluidine blue (10 min stain)

Literature:

1. el-Zimaity, Hala & Wu, J & Graham, DY. (1999). Modified Genta triple stain for identifying Helicobacter pylori. Journal of clinical pathology. 52. 693-4. 10.1136/jcp.52.9.693.

2. Laine L, Lewin DN, Naritoku W, Cohen H. Prospective comparison of H&E, Giemsa, and Genta stains for the diagnosis of Helicobacter pylori. Gastrointest Endosc. 1997 Jun;45(6):463-7. doi: 10.1016/s0016-5107(97)70174-3. PMID: 9199901.

3. Leung, K., & Gibbon, K.J., (1996) A rapid staining method for Helicobacter pylori in gastric biopsies Journal of Histotechnology, v. 19, pp. 131

4.