Acid Fast Stained Bacteria @1000xTM. Acid-fast Bacteria Stain Hot Pink. NonAcid-fast Stain Purple.

Acid-fast Ziehl Neelsen Stain Reaction 
Differential Test to Identify Mycobacterium & Nocardia Bacteria 
LAB NOTES from Science Prof Online


Many bacteria of the genera Mycobacterium and Nocardia are medically significant, causing infectious diseases such as tuberculosis, leprosy and other lung and skin infections. Therefore, it is clinically important to be able to accurately identify members of these genera. The acid-fast stain is a first step in identification of these types of bacteria.

Article Summary: Acid-fast staining is used to identify bacteria of the genera Mycobacteria & Nocardia, which have mycolic acid in their cell wall. Here's how it works.
Acid-fast Ziehl-Neelsen Stain Reaction
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Acid-fast Stain Reaction Explained
Primary stain: The hot pink appearance of Acid-fast cells is caused by Carbol fuchsin, the primary (first) stain, which is driven into acid-fast cells using the heat from a water bath.

Decolorizer: This step does not remove the Carbol fuchsin stain trapped within the waxy, acid-fast cell wall, but does remove the stain from bacterial cells that do not have wax in their cell wall.

Secondary stain (counterstain): The crystal violet counterstain imparts purple color to the colorless nonacid-fast bacteria, but doesn't change the color of acid-fast cells.​
After this staining procedure, the Acid-fast cells will appear pink because the primary stain, Ziel’s carbol fuschion, has been driven into the bacteria’s waxy cell wall with the heat from the water bath. Acid-fast cells also typically clump together, due to the wax in their cell wall. The Nonacid-fast cells (bacteria that do not have a waxy cell wall) will appear purple, having retained the counterstain, crystal violet, after the primary stain was removed by the decolorizer.

Application of Primary Stain: 1. Carbol fuchsin primary stain of acid-fat stain; 2. Carbol fuchsin being applied to slide that had been prepared with acid-fast controls and an unknown bacteria. Blotting paper has been put on top of the slide. Then the blotting paper is saturated with stain and heated over water bath; 3. Clothes pins are useful for handling the slide; 4. Blotting paper is discarded and slide is rinsed. 
Application of Decolorizer: 1. Acid alcohol decolorizer for acid-fast stain; 2. Drizzle decolorizer down slide for 10 - 15 seconds, while watching to see that stain is removed from negative control; 3. Rinse.
Application of Counterstain: 1. Secondary stain (coounterstain), crystal violet; 2. Crystal violet is applied to slide and left for one minute; 3. Rinse; 4. Stained acid fast slide, with + control on left, unknown in center and - control on right.  Go to > More acid-fast stain photos.
Acid-fast bacteria Mycobacterium smegmatis viewed under oil immersion @ 1000xTM
Non-acid-fast bacteria Staphylococcus epidermidis viewed under oil immersion @ 1000xTM
Acid-fast bacteria Mycobacterium smegmatis viewed under oil immersion @ 1000xTM
Non-acid-fast bacteria Staphylococcus epidermidis viewed under oil immersion @ 1000xTM
SPO Video: How to Prepare a 
Bacterial Smear for 
Acid Fast (Ziehl-Neelsen) Stain
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Page last updated: 2/2015
Photographic Guide to Completing the Acid-fast Stain​
Double click on photo strip for a slideshow of larger images.
SPO Video: How to Do an Acid-fast 
Ziehl-Neelsen Stain

Microbiology Lab Videos
Instructor's Corner
Cell Wall of Mycobacteria and Nocardia
The Gram stain is a differential stain reaction that is used to categorize most bacteria as either Gram+ or Gram-. This distinction is due to differences in the bacterial cell wall structure of these two categories of bacteria and has significance with respect to which antibiotics can be used to kill or control bacteria.

Bacteria of the genera Mycobacterium and Nocardia have unusual cell walls that are waxy and nearly impermeable due to the presence of the waxy molecule mycolic acid. Bacteria that produce mycolic acid are highly resistant to disinfectants, desiccation and are difficult to stain with water-based stains such as the Gram.

Because the cell wall is resistant to water-based stains, acid-fast organisms require a special staining technique involving heat to drive stain into their waxy cell wall.

Heat Fixing a Bacteria Smear 
Prior to staining bacteria, a bacterial smear must be heat fixed onto a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.

Steps of Acid Fast Staining Procedure
The protocol for staining acid-fast organisms is as follows (see photos below):
  • Place a strip of blotting paper over the slide.
  • Place the slide over a screened water bath and then saturate blotting paper with primary stain Ziehl’s carbol fuschion.
  • Allow slide to sit over water bath for 3 – 5 minutes. Reapply stain if it begins to dry out.
  • Discard blotting paper and rinse slide until water runs clear.
  • Flood slide with decolorizer, Acid Alcohol, for 10 – 15 seconds and then rinse.
  • Flood slide with counterstain, Crystal Violet, for one minute and then rinse.

Acid-fast Bacterial Smear and Stain Videos
Virtual Microbiology

You have free access to a large collection of materials used in two college-level introductory microbiology courses (8-week & 16-week). The Virtual Microbiology Classroom provides a wide range of free educational resources including PowerPoint Lectures, Study Guides, Review Questions and Practice Test Questions.
Prokaryotic Cell, Mariana Ruiz

Photographic guides to differential stains 
now available!
1. Gram
2. Acid-fast
3. Endopsore

Sources and Resources
  • Schauer, Cynthia (2007) Lab Manual to Microbiology for the Health Sciences, Kalamazoo Valley Community College.
  • Bauman, R. (2014) Microbiology with Diseases by Taxonomy 4th ed., Pearson Benjamin Cummings..

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