Ziehl-Neelsen staining
The Ziehl-Neelsen stain (after Franz Ziehl and Friedrich Neelsen ) is a common stain for microscopic specimens in microbiology in order to add so-called “acid-fast” bacteria (e.g. mycobacteria and nocardia ) by staining other non- “acid-fast” bacteria distinguish. This contrast staining is an important differential diagnostic aid for the identification of certain pathogens, especially the pathogens of tuberculosis and leprosy. An allocation to a genus or a species of bacteria is not possible with this characteristic of acid resistance alone.
The staining principle is based on the fact that these bacteria in particular contain special lipids ( waxes , mycolic acids ) in the cell wall , which prevent the bacteria from being stained by other usual staining processes by preventing the hydrophilic dye from penetrating . In Ziehl-Neelsen staining, carbolic fuchsine (phenol fuchsine) is used to stain in the heat, so that on the one hand the dye penetrates despite the lipid shell, but on the other hand the dye is difficult to extract from the bacteria using conventional decolorizing methods at normal temperature. The objects that are not “acid-resistant” are then discolored again with hydrochloric acid or a mixture of alcohol and hydrochloric acid at normal temperature. Only the "acid-fast" bacteria retain the dye during this treatment and therefore remain colored red, all other bacteria lose the dye again.
A coloration based on the same principle is the so-called auramine-rhodamine coloration . A fluorescent dye ( auramine O ) is used instead of fuchsine. Like the fuchsin, this remains in the bacteria during the discoloration and cannot be removed in this way either. Under the fluorescence microscope , the “acid-fast” bacteria then glow orange and stand out clearly from the background.
The examination of histological specimens and - in the case of tuberculosis of the lungs - of sputum specimens with these stains is relatively time-consuming, since one has to use a microscope with high magnification and often only very few mycobacteria can be found, which are sufficient for diagnosis. The method of staining and microscopic examination is still a quick way of identifying the pathogen in tuberculosis. However, the gold standard is still the culture, which for Mycobacterium tuberculosis takes a few weeks because of its slow reproduction.
method
The heat-fixed bacterial smears or histological specimens are coated with an aqueous-alcoholic carbol fuchsin solution (phenol fuchsin) and heated three times over a glowing Bunsen burner flame until they vaporize (not boiling!). The dye penetrates into all bacteria, including through the lipid-containing cell walls into the mycobacteria or other "acid-fast" bacteria. The dye is then poured off and briefly rinsed off with tap water. Then it is decolorized with alcohol, which contains 3% hydrochloric acid, for about one minute, whereby the "acid-fast" bacteria retain the color and only the objects that are not “acid-resistant” are decolored. After briefly rinsing the solution under tap water, the objects that are not “acid-resistant” are usually counter-stained with 0.3 to 1 percent methylene blue solution (about three minutes). In a modified procedure, Janus green B is used as a counterstain. After counterstaining, it is rinsed with water.
Acid-fast bacteria are then colored red, while everything else only absorbs the dye of the counterstaining (here blue).
Historical
Paul Ehrlich is considered the discoverer of the phenomenon of acid-resistant rods in 1882 , he colored with crystal violet . Franz Ziehl (addition of phenol, 1882) and Friedrich Neelsen (use of fuchsin, 1883) improved this process by using a different dye solution, beef in 1883 by heating it during dyeing, which is where the name comes from.
The method for testing acid resistance using auramine staining comes from P. Hagemann (1938).
Individual evidence
- ↑ P. Varughese, DM Helbecque, KB McRae, L. Eidus: Comparison of strip and Ziehl-Neelsen methods for staining acid-fast bacteria. In: Bulletin of the World Health Organization. Volume 51, Number 1, 1974, pp. 83-91, ISSN 0042-9686 . PMID 4141944 . PMC 2366253 (free full text).
- ↑ LC Scherer, RD Sperhacke, ML Rossetti, A. Ruffino-Netto, AL Kritski: Usefulness of the polymerase chain reaction dot-blot assay, used with Ziehl-Neelsen staining, for the rapid and convenient diagnosis of pulmonary tuberculosis in human immunodeficiency virus -seropositive and -seronegative individuals. In: Infectious disease reports. Volume 3, number 1, March 2011, p. E3, ISSN 2036-7430 . doi : 10.4081 / usually 2011.e3 . PMID 24470902 . PMC 3892596 (free full text).