What is Ziehl-Neelsen staining?

• Originally created by Ziehl and then improved by Neelsen, the Ziehl-Neelsen staining method is a differential staining method; therefore, the name Ziehl-Neelsen stain.
• Neelsen created the Ziehl-Neelsen Staining Technique by heating carbol-fuschin from Ziehl’s experiment, adding a decolorizing agent made of acid-alcohol, and a counter stain made of methylene blue dye.
• The mechanism is called Acid-Fast Stain since it employs acid-alcohol, and the hot mechanism of Acid-Fast Staining because it employs heat. Both terms are equivalent when referring to the Ziehl-Neelsen Staining method.
• This approach is used to stain microorganisms which are hard to stain with basic stains such as negative staining or gramme staining. Mycobacterium spp. is one of the most difficult pathogens to eradicate using Ziehl-Neelsen chemicals.
• Mycolic acid, a lipoidal compound found in mycobacterium, actinomycetes, norcadia, isospora, cryptosporidium, and certain fungi, makes up the thick cell walls of these organisms.
• Mycolic acid is difficult to stain, therefore simple stains such as gramme staining cannot enter the thick cell wall of these organisms.
• They need more severe treatments, such as the Ziehl-Neelsen or hot technique of Acid-fast stain, to allow stain penetration for identification and analysis.

Objectives of the Ziehl-Neelsen Staining

• to distinguish between acid-fast and slow-growing bacteria.
• Mycobacterium species are stained.

Principle of the Ziehl-Neelsen Staining

• Basic fuchsin and phenol chemicals are employed in the Ziehl-Neelsen stain to colour Mycobacterium species’ cell walls.
• The employment of heat, carbol-fuschin, and phenol facilitates penetration of the bacterial cell wall for visibility because mycobacterium does not readily attach to simple stains.
• Mycobacterium’s cell wall is waxy, hydrophobic, and impermeable because it has a high lipid content composed of mycolic acid. These 90-carbon ß-hydroxycarboxylic acids determine how resistant the bacteria are to acids.
• Use of the basic compound carbol-fuschin causes significant binding to the bacteria’s harmful by products, such as mycolic acid and the cell wall lipids. Heat and the addition of acid alcohol combine to generate a powerful complex that is difficult to remove using solvents.
• The principal dye, carbol-fuschin, has a red hue that the acid-fast bacteria absorb.
• While non-acid-fast bacteria readily discolour when acid-alcohol is added and absorb the counterstain pigment methylene blue, making them seem blue
• Mycobacterium leprae and Mycobacterium tuberculosis have both been identified using this method.

Reagents used in the Ziehl-Neelsen Stain

1. Carbol-Fuschin (Primary dye)
2. 20% sulphuric acid or acid-alcohol (Decolorizer)
3. Methylene Blue dye (counterstain) or malachite green

Preparation of reagents

Carbol fuschin

• Distilled water- 100ml
• Basic fuschin- 1g
• Ethyl alcohol (100% ethanol)- 10ml
• Phenol crystals- 5ml

Acid alcohol (3% hydrochloric acid in 95% ethyl alcohol)

• Ethyl alcohol- 95 ml
• Distilled water- 2 ml
• Concentrated hydrochloric acid (3 mL)

0.25% methylene blue in 1% acetic acid.

• Methylene blue-0.025g
• Distilled water- 99ml
• Acetic acid- 1ml

Procedure for Ziehl-Neelsen Staining

1. Make a smear of the sample culture on a sterile, clean microscope slide, then heat-fix the smear over blue heat.
2. Fill and load the smear with carbol fuschin, and afterwards gradually heat it till fumes begin to emit.
3. After letting, it sits for five minutes, rinse it off with softly running tap water.
4. Add 20% sulfuric acid, then wait one to two minutes. Continue doing this until the smearing becomes pink.
5. Remove the acid with water.
6. Fill the dish to the brim with methylene blue dye, let it sit for two to three minutes, then rinse with water.
7. Dry the stain naturally before checking it with an oil immersion lens.

Results and Interpretation

• Pink stains are produced when acid-fast bacteria retain the main colourant, carbol-fuschin.
• Methylene blue is absorbed by non-acid fat bacteria, which makes them appear blue.

Applications of Ziehl-Neelsen Staining

• used to examine and classify different Mycobacterium species.
• It is used to distinguish between bacilli that grow quickly and those that don’t.
• It is used to identify various fungus species, including Cryptosporidium.

Limitations of Ziehl-Neelsen Staining

• It only works to distinguish acid-fast bacilli.
• The organism’s physical form is distorted.

References and Sources

• 2% – https://labhelpline.com/2019/05/23/ziehl-neelsen-staining-technique-for-acid-fast-bacilli/
• 1% – https://www.uwyo.edu/virtual_edge/units/acidfast_stain.html
• 1% – https://www.ncbi.nlm.nih.gov/pubmed/17533853/
• 1% – https://www.differencebetween.com/difference-between-acid-fast-and-vs-non-acid-fast-bacteria/
• 1% – https://www.carolina.com/specialty-chemicals-a/acid-alcohol-3-hydrochloric-acid-in-95-ethanol-laboratory-grade-500-ml/841733.pr
• 1% – http://whocctblab.fondazionesanraffaele.it/uploads/2/0/8/2/20828554/_ios_ebp-ziehl-neelsen_staining.pdf
• <1% – https://www.researchgate.net/post/Why_Are_Mycobacteria_Acid_Fast
• <1% – https://quizlet.com/145783763/microbiology-hw-quiz-flash-cards/
• <1% – https://en.wikipedia.org/wiki/Kinyoun_stain
• <1% – https://en.wikipedia.org/wiki/Acid-fastness