Ziehl–Neelsen stain

The Ziehl- Neelsen stain (after Franz Ziehl and Friedrich Neelsen ) is a conventional staining for microscopic preparations in microbiology, so-called " acid-fast " bacteria (eg mycobacteria and Nocardia ) by staining of other non- " acid-resistant " bacteria to differ. This counterstain is an important differential diagnostic aid for the identification of certain pathogens, especially pathogens of tuberculosis and leprosy. An assignment to a genus or a species of bacteria is with this aspect of the acid strength alone is not possible.

The coloring principle is based on that, for these particular bacteria in particular of the cell wall lipids ( waxes, mycolic acids ) are included to prevent with conventional dyeing processes other that the bacteria are stained, they prevent the penetration of the hydrophilic dye. The Ziehl- Neelsen stain ( Phenolfuchsin ) is colored in the heat with carbol fuchsin, so that on the one hand, the dye despite the lipid envelope penetrates, but on the other hand, the dye with usual Entfärbemethoden is difficult to extract again from the bacteria at normal temperature. Thereafter, the non- " acid-resistant " properties are decolorized with hydrochloric acid or a mixture of alcohol and hydrochloric acid at normal temperature. Only the " acid-resistant " bacteria maintain this treatment the dye and remain colored red, all other bacteria lose the dye again.

A based on the same principle is the so-called coloring auramine - rhodamine stain. Here, a fluorescent dye ( auramine O) is used instead of fuchsin. This remains in decolorizing as the fuchsin in the bacteria and can no longer detach themselves well in this way. Under the fluorescence microscope, then light the " acid-resistant " bacteria orange and thus cancel clearly from the background.

The examination of histological preparations, and - in pulmonary tuberculosis - sputum preparations with these dyes is relatively time consuming because you have the microscope with high magnification and often very few mycobacteria are found, however, sufficient for diagnosis. The method of staining and microscopy provides in tuberculosis but still a quick way of finding pathogens dar. but the gold standard is still the culture, which will take several weeks for Mycobacterium tuberculosis due to its slow propagation.

Method

The heat- fixed bacterial smears or histological specimens are covered with aqueous-alcoholic carbol fuchsin ( Phenolfuchsin ) and a luminous Bunsen burner flame three times until steaming (not boiling! ) Heated. Here, the dye penetrates into all bacteria, even through the lipid -containing cell walls in the mycobacteria or other " acid-resistant " bacteria. The dye is then poured off and rinsed briefly in tap water. Then, about one minute with a alcohol containing 3% hydrochloric acid and decolorized, but with the " acid-resistant " bacteria retain the dye, and not only the " acid-resistant " properties are discolored. After briefly rinsing the solution with tap water is usually a counterstain is not " acid-fast " objects with 0.3 - to 1 -percent methylene blue solution ( about three minutes). In a modified process, Janus Green B is used as counterstain. After counterstaining is rinsed with water.

Acid-fast bacteria are then dyed red, while everything else receives only the dye counter-staining (blue here).

Historical

As a discoverer of the phenomenon of acid- fast bacilli in 1882 applies Paul Ehrlich, he colored with crystal violet. Franz Ziehl ( addition of phenol, 1882) and Friedrich Neelsen (use of fuchsin, 1883) have this procedure improved by using a different dye solution, beef in 1883 by heating in dyeing.

The method of testing for acid strength by the auramine staining comes from P. Hagemann (1938).

• Histological staining
• Microbiological test method