Ziehl–Neelsen stain
Ziehl-Neelsen staining is a type of Acid-fast stain, first introduced by Paul Ehrlich. Ziehl–Neelsen staining is a bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria.  It is named for two German doctors who modified the stain: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898).

Mycobacteria
The genus Mycobacterium is a slow growing bacteria, made up of small rods that are slightly curved or straight, and are considered to be gram positive. Some types of Mycobacteria form branches or filaments. Some mycobacteria are free-living saprophytes, but many are pathogens that cause disease in animals and humans. Mycobacterium bovis causes tuberculosis in cattle. Since tuberculosis can be spread to humans, milk is pasteurized to kill any of the bacteria.Some Mycobacteria species that cause disease in humans include Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium bovis, Mycobacterium africanum and members of the Mycobacterium avium complex. Mycobacterium tuberculosis is a species of Mycobacterium that causes tuberculosis (TB). Mycobacterium tuberculosis is an airborne bacterium that typically infects the human lungs. Symptoms of TB include a bad cough, chest pain, fatigue, weight loss, no appetite, chills, fever and night sweats. The typical regimen for treating a Latent TB infection includes the use of isoniazid, rifapentine, and rifampin. The regimen is changed for those who have developed a drug resistant strain of TB. Testing for TB includes blood testing, skin tests, and chest x-rays. When looking at the smears for TB, it is stained using and acid-fast stain. These Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia. The reagents used for Ziehl–Neelsen staining are – carbol fuchsin, acid alcohol, and methylene blue. Acid-fast bacilli are bright red after staining.

Fungi
Ziehl-Neelsen staining is a type of narrow spectrum fungal stain. Narrow spectrum fungal stains are selective, and they can help differentiate and identify fungi. The results of ZIehl- Neelsen staining is variable because many fungal cell walls are not acid fast. An example of a common type of acid-fast fungus that is usually stained with Ziehl-Neelsen staining is called Histoplasma (HP). Histoplasma is found in soil and the feces of birds and bats. Humans can contract histoplasmosis by inhalation of the fungal spores. Histoplasma enters the body and goes to the lungs where the spores turn into yeast. The yeast gets into the blood stream and affects lymph nodes and other parts of the body. Usually people do not get sick from inhaling the spores, but if they do they usually have flu like symptoms. Another variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula. Some free endospores can be confused with small yeasts, so staining is used to identify the unknown fungi. It is also useful in the identification of some protozoa, namely Cryptosporidium and Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in sputum sample for ovum and parasite (O&P) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of paragonimiasis closely resemble those of TB.

History
In 1882 Robert Koch discovered the etiology of tuberculosis. Soon after Koch’s discovery, Paul Ehrlich developed a stain for mycobacterium tuberculosis, called the alum hematoxylin stain. Franz Ziehl then altered Ehrlich’s staining technique by using carbolic acid as the mordant. Friedrich Neelsen kept Ziehl’s choice of mordant but changed the primary stain to carbol fuchsin. Ziehl and Neelsen’s modifications together have developed the Ziehl-Neelsen stain. Another acid-fast satin was developed by Joseph Kinyoun by using the Ziehl-Neelsen staining technique but removing the heating step from the procedure. This new stain from Kinyoun was named the Kinyoun stain.

Procedure
A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. The Ziehl-Neelsen method of staining for TB is known as the “hot plate” method which means the Ziehl-Neelsen method uses bright field microscopy.  To begin the staining process, a bacterial smear must be done. The smear should be evenly spread across the center of the slide. The smear is covered in a piece of bibulous paper and the smear is stained with carbol fuchsin. The slide is heated for five minutes while keeping the bibulous paper moist with the carbol fuchsin. The bibulous paper is removed and the slide is rinsed with distilled water. Acid-alcohol is used to decolorize the slide until the runoff is clear. The decolorizer removes the stain from non-acid-fast cells. The slide is rinsed with distilled water to insure all of the decolorizer is off of the slide. The slide is stained with the counter stain of methylene blue for one minute. Rinse the slide with distilled water. Blot, do not rub, the slide dry in a tablet of bibulous paper. When the slide is dry observe the bacteria under a microscope with oil immersion.

Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB culture should be performed along with an AFB stain; this has a much higher negative predictive value.

Mechanism explanation 
Initially, carbol fuchsin stains every cell. When they are de-stained with acid-alcohol, only non-acid-fast bacteria get de-stained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counter stain is applied, non-acid-fast bacteria pick it up and become blue (methylene blue) or green (malachite green) when viewed under the microscope. Acid-fast bacteria retain carbol fuchsin so they appear red.

Modifications
1% sulfuric acid alcohol for actinomycetes, nocardia.
0.5–1% sulfuric acid alcohol for oocysts of isospora, cyclospora.
0.25–0.5% sulfuric acid alcohol for bacterial endospores.
Differential staining – glacial acetic acid used, no heat applied, secondary stain is Loeffler's methylene blue.
 Kinyoun modification (or cold Ziehl–Neelsen technique) is also available.
 A protocol in which a detergent is substituted for the highly toxic phenol in the fuchsin staining solution.